Shin JB et al. (2005), Xenopus TRPN1 (NOMPC) localizes to microtubule-...

XB-ART-1469

Proc Natl Acad Sci U S A 2005 Aug 30;10235:12572-7. doi: 10.1073/pnas.0502403102.

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Xenopus TRPN1 (NOMPC) localizes to microtubule-based cilia in epithelial cells, including inner-ear hair cells.

Shin JB , Adams D , Paukert M , Siba M , Sidi S , Levin M , Gillespie PG , Gründer S .

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In vertebrates, the senses of hearing and balance depend on hair cells, which transduce sounds with their hair bundles, containing actin-based stereocilia and microtubule-based kinocilia. A longstanding question in auditory science is the identity of the mechanically sensitive transduction channel of hair cells, thought to be localized at the tips of their stereocilia. Experiments in zebrafish implicated the transient receptor potential (TRP) channel NOMPC (drTRPN1) in this role; TRPN1 is absent from the genomes of higher vertebrates, however, and has not been localized in hair cells. Another candidate for the transduction channel, TRPA1, apparently is required for transduction in mammalian and nonmammalian vertebrates. This discrepancy raises the question of the relative contribution of TRPN1 and TRPA1 to transduction in nonmammalian vertebrates. To address this question, we cloned the TRPN1 ortholog from the amphibian Xenopus laevis, generated an antibody against the protein, and determined the protein's cellular and subcellular localization. We found that TRPN1 is prominently located in lateral-line hair cells, auditory hair cells, and ciliated epidermal cells of developing Xenopus embryos. In ciliated epidermal cells TRPN1 staining was enriched at the tips and bases of the cilia. In saccular hair cells, TRPN1 was located prominently in the kinocilial bulb, a component of the mechanosensory hair bundles. Moreover, we observed redistribution of TRPN1 upon treatment of hair cells with calcium chelators, which disrupts the transduction apparatus. This result suggests that although TRPN1 is unlikely to be the transduction channel of stereocilia, it plays an essential role, functionally related to transduction, in the kinocilium.

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Species referenced: Xenopus laevis
Genes referenced: actb actl6a ank1 cdh23 mcoln3 naa50 nos1 pkd2 trpa1 trpc1 trpc3 trpm2 trpm3 trpv1 trpv4 tuba4b XB5813854
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	Fig. 1. Localization of TRPN1 in lateral-line hair cells of stage-45 Xenopus embryos. (A and B) Dissecting microscope dorsal view of embryos that had been stained with FM1-43 to label lateral-line hair cells. In A, red arrows point to lateral-line hair cells around the eye; in B, white arrows highlight the absence of FM1-43 staining around the eyes of neomycin-treated embryos. (C and D) Shown are A and B merged with their respective transmitted light images to better show the localization of FM1-43 staining. (E) Staining with the anti-TRPN1 antibody. Dorsal view of an embryo that had been bleached to distinguish alkaline phosphatase staining from pigment. Red arrows point to staining of the lateral-line stitches around the eye. Blue arrows point to staining of the nostrils. (F) Anti-TRPN1 staining of unbleached, neomycin-treated embryo. Nostrils are positive for TRPN1, but staining around the eyes is absent, showing that the neomycin effect is specific to lateral-line hair cells. (G) Close-up of the lateral-line staining surrounding the eye shown in E. Red arrows point to signal in lateral-line stitches. (H) Dorsal view of bleached embryo. Preadsorption of the TRPN1 antibody with the epitope peptide prevented TRPN1 staining of both the lateral line and the nostril (white arrows).
	Fig. 2. Localization of TRPN1 in cilia of epidermal cells. (A) Bleached tail from a control tadpole. Preadsorption of the anti-TRPN1 antibody with the epitope peptide prevented TRPN1 staining in the epidermis of embryos. (B) Staining of epidermal cells by the anti-TRPN1 antibody (dark blue spots). Full panel width is â‰ˆ600 Î¼m. (C) Planar sections through epidermal tissue showing TRPN1 staining in open circles, consistent with the predicted cell-membrane localization of TRPN1. Full panel width is â‰ˆ600 Î¼m. (D-F) Fluorescence of doubly labeled tadpole tails. Maximal projections of z-series. (D) TRPN1 (FITC). (E) Î±-Tubulin (TRITC). (F) Merged images. TRPN1 stain is seen in bright spots (e.g., green arrow); green hue in muscle in D is autofluorescence. Î±-Tubulin is brightest in neurons and ciliated cells (e.g., red arrow). The merged image shows that the TRPN1 and Î±-tubulin are colocalized in the ciliated cells (yellow arrow). (G-J) Higher-magnification images of the cilia of epidermal cells. (G) Transmitted light image, single section from confocal z-series showing the cilia. (H) TRPN1. (I) Î±-Tubulin in the cilia shown in G, maximum projection of z-series. (J) H and I merged, showing colocalization of TRPN1 and tubulin in cilia. Blue box indicates approximate boundaries of high magnification shown in M. (K) Projection of three proximal sections from J. (L) Projection of three sections from J showing the distal ends of cilia. Blue arrows point to some of the visible cilia tips; both the proximal and the distal ends of cilia stain more heavily for TRPN1. (M) Very high magnification of some cilia distal tips, corresponding to the box in J. The z-series used to create J was restored by using Wiener deconvolution, a process that removes artifactual light from images collected at this high magnification. PHOTOSHOP (Adobe Systems, San Jose, CA) was then used to increase the resolution (bicubic) and increase contrast. Blue arrows point to the tips where TRPN1 is concentrated. Punctate TRPN1 staining is also visible along the cilia. (Scale bars: 200 Î¼m, D-F; 8 Î¼m, G-J.) (Full width: 70 Î¼m, K-L; 20 Î¼m, M.)
	Fig. 3. Localization of TRPN1 in Xenopus vestibular hair cells. (A) Saccular hair cells in profile. Triple staining for TRPN1 (green), actin (red), and tubulin (blue); pseudocolor code also applies to B, E, F, K, and O. Arrows indicate TRPN1 at the kinocilial bulb. (B) Preincubation of the TRPN1 antibody with the epitope peptide prevents TRPN1 staining at the kinocilia tip. (C and D) Cdh23 (green), actin (red), and tubulin (blue) in hair cell under control conditions. Cdh23 is present in the two to six tallest stereocilia in a stripe adjacent to the kinocilial bulb. (E) TRPN1 and actin in hair cell under control conditions. Note TRPN1 in kinocilia bulb (arrow). (F) After EGTA treatment (5 mM, 30 min), the TRPN1 signal appears at the base of the kinocilium (arrow). (G) Graphic illustration of the optical section in the confocal sections of H-O. (H-O) Whole-mount view of hair bundles, in a slightly angular optical section as illustrated in G. (H-K) Control conditions. (H) Actin. (I) TRPN1. (J) Tubulin. (K) Merged signals. (L-O) After EGTA treatment. (L) Actin. (M) TRPN1. (N) Tubulin. (O) Merged signals. Full panel widths: 24 Î¼m, A and B; 8 Î¼m, C; 25 Î¼m, D; 13 Î¼m, E and F; 45 Î¼m, H-O.
	Fig. 4. Sequence comparison of TRPN orthologs. Sequence alignment of xlTRPN1 with TRPN1 from zebrafish (DrTRPN) and Drosophila (DmTRPN). Amino acids showing a high degree of identity are shown as white letters on a black background. Putative transmembrane domains and P-loop are indicated by bars; ankyrin repeats are indicated by hatched bars; consensus sites for phosphorylation by protein kinase C are indicated by an asterisk; and a consensus site for N-linked glycosylation is indicated by a branched symbol. The conserved TRP-box after S6 is indicated. Transmembrane domains were predicted for xTRPN with the help of the TMPRED program (available at www.ch.embnet.org); transmembrane domains S2 and S3 could not be unequivocally assigned. GenBank accession nos. are AY313897 (DrTRPN) and AF242296 (DmTRPN). The Drosophila sequence shown is the TRP box-containing splice variant of fly TRPN1 (1). 1. Sidi, S., Friedrich, R. W. & Nicolson, T. (2003) Science 301, 96-99.
	Fig. 5. Evolutionary relationship of xlTRPN1 to other TRP channels. TRPN1 channels were aligned with members of each of the TRPA, TRPC, TRPM, TRPML, TRPP, and TRPV subfamilies from Drosophila and various vertebrate species. Only transmembrane domains S4-S6 of each protein were aligned. The alignment was done with CLUSTALX, and the phylogram was established by maximum-likelihood analysis using the software TREE-PUZZLE. The tree was then rooted in TREE-VIEW with TRPA and PAINLESS as an outgroup. Support values of internal branches that were <90% are indicated. Length of the branches indicates evolutionary distance. The following cDNAs or genes were used for the alignment: Mm-ANKTM1 (GenBank accession no. AY231177, amino acids aligned: 835-963); Dm-PAINLESS (GenBank accession no. AY268106, amino acids: 588-734); Rn-TRPV1 (GenBank accession no. AF029310, amino acids: 537-682); Gg-TRPV4 (GenBank accession no. AF261883, amino acids: 561-704); Dm-NAN (GenBank accession no. AY262004, amino acids: 582-718); Dm-CG4536 (GenBank accession no. CG4536, amino acids: 543-675); Dm-CG8743 (GenBank accession no. CG8743, amino acids: 442-569); Mm-TRPML3 (GenBank accession no. AY083531, amino acids: 374-501); Hs-Dm-CG6504 (GenBank accession no. CG6504; amino acids: 648-769); Hs-TRPP2 (GenBank accession no. U50928; amino acids: 559-680); TRPC1 (GenBank accession no. X89066; amino acids: 465-611); Mm-TRPC3 (GenBank accession no. AF190645; amino acids: 527-658); Dm-TRP (GenBank accession no. M34393; amino acids: 508-661); Hs-TRPM2 (GenBank accession no. AB001535, amino acids: 896-1048); Dm-CG16805 (GenBank accession no. CG16805; amino acids: 922-1056); Hs-TRPM3 (GenBank accession no. AJ505026; amino acids: 772-808 and 970-1112); Xl-TRPN (amino acids: 1249-1394); Dr-TRPN1 (amino acids: 1331-1468); Dm-TRPN1 (amino acids: 1402-1557). Dm, Drosophila melanogaster; Dr, Danio rerio; Hs, Homo sapiens; Mm, Mus musculus; Rn, Rattus norvegicus; Gg, Gallus gallus.
	Fig. 6. Immunoblot analysis of xlTRPN1. Immunoblot with the antibody against the C terminus of xlTRPN1. Left lane, uninjected oocytes. Right lane, oocytes injected with cRNA coding for full-length xlTRPN1 (two oocytes per lane). Note the presence of xlTRPN1 in uninjected oocytes.
	unnamed (putative transient receptor potential channel) (prospective names include NOMPC or TRPN1) gene expression in Xenopus laevis embryo, assayed via immunohistochemistry, NF stage 45, dorsal view, anterior up.

References [+] :

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