Bechstedt S et al. (2010), A doublecortin containing microtubule-associate...

ECB-ART-50166

Nat Commun 2010 Apr 12;1:11. doi: 10.1038/ncomms1007.

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A doublecortin containing microtubule-associated protein is implicated in mechanotransduction in Drosophila sensory cilia.

Bechstedt S , Albert JT , Kreil DP , Müller-Reichert T , Göpfert MC , Howard J .

Abstract
Mechanoreceptors are sensory cells that transduce mechanical stimuli into electrical signals and mediate the perception of sound, touch and acceleration. Ciliated mechanoreceptors possess an elaborate microtubule cytoskeleton that facilitates the coupling of external forces to the transduction apparatus. In a screen for genes preferentially expressed in Drosophila campaniform mechanoreceptors, we identified DCX-EMAP, a unique member of the EMAP family (echinoderm-microtubule-associated proteins) that contains two doublecortin domains. DCX-EMAP localizes to the tubular body in campaniform receptors and to the ciliary dilation in chordotonal mechanoreceptors in Johnston's organ, the fly's auditory organ. Adult flies carrying a piggyBac insertion in the DCX-EMAP gene are uncoordinated and deaf and display loss of mechanosensory transduction and amplification. Electron microscopy of mutant sensilla reveals loss of electron-dense materials within the microtubule cytoskeleton in the tubular body and ciliary dilation. Our results establish a catalogue of candidate genes for Drosophila mechanosensation and show that one candidate, DCX-EMAP, is likely to be required for mechanosensory transduction and amplification.

PubMed ID: 20975667
PMC ID: PMC2892299
Article link: Nat Commun
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	Figure 1. DCX-EMAP, a doublecortin-domain-containing member of the echinoderm–microtubule-associated protein family.(a) The exon–intron structure of CG42247 showing the piggyBac (f02655) insertion into the intron between exons 4 and 5. (b) The domain structure of the DCX-EMAP protein. Two (one for the truncated form) N-terminal doublecortin domains (DCX) are followed by a HELP domain. The C terminus of the 819 aa protein consists of 10 WD40 domains, shown in grey. (c) The EMAP family tree (S. purpuratus, C. elegans, D. melanogaster, D. rerio) and the five mammalian (mouse EML 1-5) EMAP homologues are shown (similarity matrix, blosum; parameters: neighbour joining; tie breaking, systematic). (d–f) U2OS cells expressing α-tubulin-mCherry (d) transfected with DCX-EMAP-EGFP (e), and overlay (f) showing co-localization between microtubules and DCX-EMAP and large microtubule bundles not observed in non-transfected cells (scale bar, 20 μm).
	Figure 2. Mutations in DCX-EMAP are associated with uncoordination and deafness in flies.(a) Flight initiation assay37. (b) Results from the flight initiation assay displayed as box plots. The box marks the 25th and 75th percentile and the median (middle line). The whiskers mark the 10th and 90th percentile. Triple asterisk, P<0.001; unpaired t-test compared with wild type; n=27 (Df(3L)BSC441/piggyBac), 41 (Df(3L)ED217/piggyBac), 51 (pBac/pBac), 46 pBac/TM3), 34 (excision), 84 (minos/minos), 118 (wild type). (c) Force step (lower trace)-evoked CAP responses (middle traces) are absent in the antennal nerves of f02655 DCX-EMAP mutants (blue), whereas robust CAP responses can be recorded from wild-type (grey) and f02655 excision controls (green). Upper panel: on step actuation, the movement of the antennal receivers of controls, but not mutants, displays the characteristic overshoot that associates with the opening of transduction channels. (d) Unstimulated fluctuations of the antennal receivers of f02655 DCX-EMAP mutants and controls. Colour code as in (c).
	Figure 3. Transduction and amplification are impaired in DCX-EMAP mutants.(a) Power spectra of unstimulated receiver vibrations in the DCX-EMAP mutant (blue traces in all panels), wild-type (grey traces) and f02655 excision controls (green). (b) Energy gain provided by active amplification deduced from the power spectra in (a)49. The range of wild-type values (one standard deviation around the mean) is marked in grey. Error bars display one standard deviation. (c) Response to pure-tone stimuli. Upper panel: Displacement response of the antennal receiver versus stimulus particle velocity. Lower panel: Mechanical sensitivity of the receiver plotted against particle velocity. Wild-type: grey, f02655 mutant: blue circles, f02655 excision control: green circles. (d) Response to force steps. Upper left panel: Displacement response of the receiver as a function of the stimulus force. Lower panel: The corresponding slope stiffness drops for small force amplitudes in wild type (grey) and excision controls, whereas it is constant for DCX-EMAP f02655 mutants (blue). The displacement-force relations of excision and control flies and the corresponding slope stiffnesses are well described by fits of a symmetric gating-spring model38. Right panel: CAP responses and predicted excess open probability versus receiver displacement. For wild type (grey) and excision controls (green), the mechanically evoked CAP response closely follows the excess open probability predicted from displacement data using a symmetric gating-spring model (solid lines for pO(×peak), dashed lines for pO(-×peak)7).
	Figure 4. The ultrastructure of the tubular body in f02655 campaniform receptors in the Drosophila haltere is altered.(a) Schematic drawing of the campaniform receptor. The tubular body of the receptor is marked and illustrated separately (right panel). (b, c) Electron micrographs of the sensory dendrite in wild-type campaniform receptors in the haltere. The distal part of the tubular body shows characteristic electron-dense material. (d, e) Electron-dense material in the distal tubular body at higher magnification. (f, g) Electron micrographs of f02655 campaniform receptors. The electron-dense material in the distal tubular body is missing. Microtubules in the proximal tubular part seem disorganized. (h, i) Distal tubular bodies of f02655 campaniform receptors at higher magnification. Scale bars represent 0.5 μm (f) and 250 nm (i).
	Figure 5. Mutant f02655 sensory neurons of Johnston's organ display an altered ultrastructure.(a) Schematic representation of Johnston's organ in sensory neurons of Drosophila antenna. The region with the ciliary dilation is marked and illustrated separately (right part). (b, c) Electron micrographs of ciliary dilations (arrowheads) in wild-type chordotonal neurons with electron-dense material in the ciliary dilation (arrows point towards regular holes and channels in the electron-dense material). (d, e) Chordotonal neuron of f02655 flies with 'empty' ciliary dilations. Note how the microtubules are still bent as in wild-type dilations. Scale bars represent 1 μm (d) and 250 nm (e), arrowheads point towards ciliary dilation.
	Figure 6. DCX-EMAP localization in campaniform receptors.(a) UAS-dsRED localization in a wing-vein campaniform receptor. (b) UAS-EMAP-GFP localizes to the tubular body and is not detectable in other cellular compartments. (c) Merge of panels a and b. Arrowhead points toward the tubular body. (d) Schematic of the campaniform receptor. (e) GFP-tubulin showing sensory cilia. (f) EMAP-dsRed localizes to ciliary dilations. (g) Merge of panels e and f (sensory cilia are marked by arrows). (h) Schematic of DCX-EMAP localization in chordotonal receptors. (i) Rhodamine-phalloidin staining the actin rods within scolopale cells. (j) UAS-IAV-GFP expressed in chordotonal neurons using DJ648-Gal4. (k) Merge showing that IAV-GFP localizes to the proximal part of the cilia. (l) Schematic of IAV localization. (m) UAS-DCX-EMAP-dsRed expressed in chordotonal neurons of Johnston's organ (n) UAS-CNN-GFP. (o) Merge of panels m and n showing the localization of DCX-EMAP-dsRed distal to CNN-GFP. (p) Schematic of DCX-EMAP-dsRed and CNN-GFP localization. All scale bars are 5 μm.

References [+] :

Albert, Mechanical signatures of transducer gating in the Drosophila ear. 2007, Pubmed