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. 2011 Jan;68(1):1-7.
doi: 10.1002/cm.20493.

NOMPC, a member of the TRP channel family, localizes to the tubular body and distal cilium of Drosophila campaniform and chordotonal receptor cells

Xin Liang  1 , Johnson Madrid, Henri S Saleh, Jonathon Howard
Affiliations
Free PMC article

NOMPC, a member of the TRP channel family, localizes to the tubular body and distal cilium of Drosophila campaniform and chordotonal receptor cells

Xin Liang et al. Cytoskeleton (Hoboken). 2011 Jan.
Free PMC article

Abstract

Mechanoreception underlies the senses of touch, hearing and balance. An early event in mechanoreception is the opening of ion channels in response to mechanical force impinging on the cell. Here, we report antibody localization of NOMPC, a member of the transient receptor potential (TRP) ion channel family, to the tubular body of campaniform receptors in the halteres and to the distal regions of the cilia of chordotonal neurons in Johnston's organ, the sound-sensing organ of flies. Because NOMPC has been shown to be associated with the mechanotransduction process, our studies suggest that the transduction apparatus in both types of sensory cells is located in regions where a specialized microtubule-based cytoskeleton is in close proximity to an overlying cuticular structure. This localization suggests a transmission route of the mechanical stimulus to the cell. Furthermore, the commonality of NOMPC locations in the two structurally different receptor types suggests a conserved transduction apparatus involving both the intracellular cytoskeleton and the extracellular matrix.

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Figures

Fig. 1
Fig. 1. Antigen information and antibody screening
A: The polypeptide with first 404 amino acids (N404) in the N-terminal of NOMPC was fused with GST and chosen as antigen. B: Western blot on the bacterial lysate with anti-NOMPC antibody (clone number: 1214-A02-01). Lane 1: uninduced cell; Lane 2: induced for 2 hours; Lane 3: induced for 4 hours. The band at ∼ 70 KD (GST-N404) was only detected at induced cell sample. C: Western blot on cell lysate of HEK 293FT cell. Lane 4: wild type HEK 293FT cell; Lane 5: HEK 293FT cell transiently transfected with N-terminal GFP-tagged full length NOMPC (NT-GFP-NOMPC). A specific band at ∼ 206 KD was detected only in transfected cell samples. D: Immunostaining on the HEK 293FT cells transfected with NT-GFP-NOMPC. Green: NT-GFP-NOMPC channel; Blue: DAPI; Red: anti-NOMPC. Only the cells show the GFP signal (NT-GFP-NOMPC, green) could be stained by anti-NOMPC antibody (red).
Fig. 2
Fig. 2. The structure of Campaniform receptor in haltere and the subcellular localization of NOMPC in campaniform receptor in the haltere
A: SEM image of a haltere and a receptor field (green in inset) in the pedicel of the haltere (pink in inset). Scale bar: 100 μm. B: High magnification SEM image of a campaniform receptor field in the haltere pedicel. C: Transmission electron microscopy image of a longitude section of a campaniform receptor in the haltere from a nompC3 fly. The whole cell region and nucleus region are highlighted in light green and light blue, respectively. It shows that campaniform receptor in nompC3 fly has a similar overall shape to that of wild type neurons [Keil,1997]. The inset shows an enlargement of the tubular body in this campaniform receptor. D: Schematic diagram of a campaniform receptor and the tubular body. Inset: the distal tip region of the campaniform receptor is clearly visualized with a mouse anti-acetylated tubulin antibody (Sigma, T6793). E: Anti-NOMPC antibody (red) exclusively stains the distal tips of tubular body, here visualized with an anti-α-tubulin antibody (green) (Abcam, Catalog No. ab15246). The inset shows a top-view of anti-NOMPC staining (red) in the distal tip of the tubular body. F: The same region of the tubular body (anti-α-tubulin, green) in the nompC3 fly did not stain with anti-NOMPC antibody. The distal tip region is not as strongly stained by the anti-α-tubulin as it is by the anti-acetylated tubulin antibody (D, inset) (Supporting Information Fig. S1).
Fig. 3
Fig. 3. Subcellular localization of NOMPC in Johnston's organ in the antenna and femoral chordotonal organ in the leg
A: Schematic picture of Johnston's organ cells. B: Anti-NOMPC antibody (red) stains the distal cilia of Johnston's organ cells (anti-α-tubulin, green). A fly strain expressing DsRed-tagged DCX-EMAP in the nervous system was used to label the position of ciliary dilation (blue). The strong red staining on the left side of the images is the strong auto-fluorescence from a piece of cuticle in this tissue section. C: The distal cilia of Johnston's organ in the nompC3 fly did not stain with anti-NOMPC antibody. To visualize the fine structure of cilia labeled with anti-α-tubulin, anti-α-tubulin signal was over-exposed (green) and phalloidin was also used to label the actin-rich scolopale (gray). D: Subcellular localization of NOMPC in leg femoral chordotonal cells. A fly strain expressing GFP in the nervous system was used to label the chordotonal cells. To visualize the fine structure of the cilia, the cytoplasmically expressed GFP signal (green) was over-exposed. We identified the ciliary dilation as the bright dot in the GFP channel (yellow), as expected from the enlargement of the cytoplasm at the dilation; furthermore, the location of the bright dot is approximately two thirds distance from basal body to the distal tip (Supporing Information Fig. S2), the expected location of the dilation based on the detailed ultrastructure [Kernan,2007]. Anti-NOMPC antibody (red) stains the distal cilia of femoral chordotonal organ cells, from the dilation to the distal tip, similar to the staining in Johnston's organ.
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