The tip-link antigen, a protein associated with the transduction complex of sensory hair cells, is protocadherin-15

Abstract

Sound and acceleration are detected by hair bundles, mechanosensory structures located at the apical pole of hair cells in the inner ear. The different elements of the hair bundle, the stereocilia and a kinocilium, are interconnected by a variety of link types. One of these links, the tip link, connects the top of a shorter stereocilium with the lateral membrane of an adjacent taller stereocilium and may gate the mechanotransducer channel of the hair cell. Mass spectrometric and Western blot analyses identify the tip-link antigen, a hitherto unidentified antigen specifically associated with the tip and kinocilial links of sensory hair bundles in the inner ear and the ciliary calyx of photoreceptors in the eye, as an avian ortholog of human protocadherin-15, a product of the gene for the deaf/blindness Usher syndrome type 1F/DFNB23 locus. Multiple protocadherin-15 transcripts are shown to be expressed in the mouse inner ear, and these define four major isoform classes, two with entirely novel, previously unidentified cytoplasmic domains. Antibodies to the three cytoplasmic domain-containing isoform classes reveal that each has a different spatiotemporal expression pattern in the developing and mature inner ear. Two isoforms are distributed in a manner compatible for association with the tip-link complex. An isoform located at the tips of stereocilia is sensitive to calcium chelation and proteolysis with subtilisin and reappears at the tips of stereocilia as transduction recovers after the removal of calcium chelators. Protocadherin-15 is therefore associated with the tip-link complex and may be an integral component of this structure and/or required for its formation.

Figure 1.

Pcdh15 splice variants. Splicing of the primary transcripts of Pcdh15 and the four isoform classes defined by the presence or absence of one of three different cytoplasmic domains. Newly discovered exons of Pcdh15 are designated with a letter suffix if located among the reported 35 exons. A dashed line indicates that one or more exons were not included in the transcript. A dotted line designates either the 3′ untranslated region (UTR) or the 5′ UTR. A signal peptide is encoded by exon 2, and a transmembrane domain (TM; brown) is encoded by exon 31. a–c, Diagrams of exon content of full-length, open reading frame-containing transcripts with one of the three alternative cytoplasmic domains encoded by exons 35 (green), 38 (blue), and 39 (pink), respectively (supplemental Fig. S1, available at www.jneurosci.org as supplemental material). Different antigens used for producing polyclonal antibodies to protocadherin-15 are shown as yellow rectangles above and below the structures of each protocadherin-15 isoform. The location of the two tryptic chicken peptides identical to human and mouse sequence that were detected by mass spectrometric analyses of purified TLA are shown as two red rectangles below the structure of protocadherin-15–CD1 (for sequence of peptides, see Table 1). The locations of the six reported Ames waltzer alleles (av-2J, av-Tg2742Rpw, av-5J, av-Jfb, av-J, and av-3J) are shown in a. Note that, for all transcripts of mouse Pcdh15 shown in a–c, there are no constant coding exons. However, within each class of Pcdh15 transcripts (CD1, CD2, and CD3), the 3′ UTR encoded by exons 35, 38, and 39, respectively, appear to be constant, but that observation may reflect an ascertainment bias because the reverse primer for each of the three classes was located in the 3′ UTRs (green, blue, and pink arrows) (see also Ahmed et al., 2003). Black arrow in exon 1 is the location of the forward primer. d, An isoform class of protocadherin-15 expressed in the mouse inner ear that is predicted to be secreted. The GenBank accession number for each isoform is in supplemental Table S3 (available at www.jneurosci.org as supplemental material).

Figure 2.

Distributions of protocadherin-15–CD1, protocadherin-15–CD2, and protocadherin-15–CD3 in the developing mouse cochlea. P2 mouse organ of Corti, 1 d in vitro. Hair bundles from the apical end of the apical coil (a, d, g), middle of the apical coil (b, e, h), and basal (c, f, i) regions of the cochlea stained with antibody PB303 to protocadherin-15–CD1 (a–c), antibody PB464-2B to protocadherin-15–CD2 (d–f), and antibody PB375 to protocadherin-15–CD3 (g–i). The left half of each panel shows the distribution of protocadherin-15, and the right half shows the merge with F-actin. I, Inner hair cell; O1, O2, O3, outer hair cells in rows 1, 2, and 3 respectively. Scale bars, 10 μm.

Figure 3.

Distribution of protocadherin-15–CD1 in the hair bundle. a–d, Confocal images of mouse hair bundles double labeled with antibody PB303 directed against protocadherin-15–CD1 (green) and phalloidin to detect F-actin (red). a, Adult mouse cochlea, inner hair cells. Arrow indicates the stereocilium shown enlarged in c. b, Adult mouse utricle. c, Detail of adult mouse inner hair cell stereocilium. d, Adult mouse utricle, detail of stereocilia. In c and d, note that the tips of the stereocilia are devoid of staining for CD1 (absence of green fluorescence above the dashes). In a–d, the left panel shows the distribution of protocadherin-15-CD1, the middle panel shows the distribution of F-actin, and the right panel shows the merge of the two stainings. Scale bars, 5 μm.

Figure 4.

Protocadherin-15–CD3 distribution in hair bundles. a–g, Confocal images of hair bundles double labeled with antibodies to protocadherin-15–CD3 (PB375 or HL5383; green) and phalloidin (red). In a–e, the left or top panel shows the distribution of protocadherin-15–CD3, the middle panel shows the distribution of F-actin, and the right or bottom panel shows the merge of the two labels. In f and g, the left panels shows protocadherin-15–CD3, and the right panel shows the merge with F-actin. a, E15 mouse ampulla, antibody PB375. b, P9 rat saccule, detail of the hair bundle, antibody PB375. c, P8 mouse utricle, detail of stereocilia tips, antibody PB375. d, Inner hair cell stereocilia bundles, mouse organ of Corti, P2 plus 1 d in vitro, antibody PB375. e, Outer hair cell stereocilia bundle, mouse organ of Corti, P2 plus 1 d in vitro, antibody PB375. f, P17 rat organ of Corti, basal turn, antibody HL5383. Note that the stereocilia of the single row of inner hair cells (top of the image, I) lack staining, whereas the V-shaped stereocilia of the three rows of outer hair cells (O1, O2, O3) have protocadherin-15–CD3 immunoreactivity in the hair bundles. g, P17 rat organ of Corti, apical turn, antibody HL5383. Note that the tips of the stereocilia in all rows within the bundle are stained. Scale bars: a, b, d–g, 5 μm; c, 1 μm.

Figure 5.

Immunogold electron microscopy of protocadherin-15–CD1 and protocadherin-15–CD3. Electron micrographs of P2 mouse utricle hair bundles stained with antibody PB303 to protocadherin-15-CD1 (a, b), non-immune rabbit IgG (c), and antibody PB375 to protocadherin-15-CD3 (d–f). Hair bundles were labeled using postembedding labeling with primary antibody followed by 10 nm gold-conjugated goat anti-rabbit Ig (a–d) or pre-embedding labeling with primary antibody followed by 5 nm gold-conjugated goat anti-rabbit Ig (e, f). f is an enlargement of the tip of the taller of the two stereocilia shown in e. Note how protocadherin-15–CD1 is excluded from the very tip of the stereocilium (a, b) and how protocadherin-15–CD3 (d–f) is distributed around the tip of each stereocilium. Scale bars, 200 nm.

Figure 6.

Distribution of protocadherin-15 ectodomain epitopes in cochlear hair bundles. a–f, P2 mouse organ of Corti, 1 d in vitro, double labeled with antibody PB473-3 (a–c, green) or antibody HL5614 (d–f, green) and phalloidin (red). The left half of each panel shows the distribution of protocadherin-15, and the right half shows the merge with F-actin. a, Apical coil, antibody PB473-3, without TX-100. b, Apical coil, antibody PB473-3, with TX-100. c, Basal coil, antibody PB473-3, with TX-100. d, Apical coil, antibody HL5614, without BAPTA. e, Apical coil, antibody HL5614, after BAPTA. f, Basal coil, antibody HL5614, after BAPTA. Kinocilia are indicated by arrows. Scale bar, 10 μm.

Figure 7.

Immunoblot analysis of the TLA and chicken protocadherin-15 splice variants. a, Western blots of immunoprecipitates obtained from Triton X-100 soluble extracts of the early posthatch chicken retina using anti-TLA mAb G19 or irrelevant control anti-hair-cell antigen mAb D10 (Richardson et al., 1990). Blots were probed with rabbit antibodies directed against the extracellular ectodomain of mouse protocadherin-15 (PB473-3), mouse protocadherin-15–CD1 (PB303), mouse serum directed against the intracellular domain of chick protocadherin-15–CD1 (M110), or rabbit antibodies directed against mouse protocadherin-15–CD3 (HL5383). Positions of Precision Markers (Bio-Rad, Hercules, CA) are indicated to the left. b–d, The exon content of full-length, open reading frame-containing transcripts with alternative cytoplasmic domains encoded by exons 31 (green), 36 (blue), and 37 (pink). These transcripts of chicken Pcdh15 are unlikely to be a comprehensive inventory. The location of tryptic peptides detected by mass spectrometric analyses of the two purified TLA bands (b) (see also Table 1) are shown as red rectangles. A GenBank accession number for each isoform is found in supplemental Table S3 (available at www.jneurosci.org as supplemental material). TM, Transmembrane domain.

Figure 8.

Colocalization of the TLA and protocadherin-15. a–c, Confocal images of the chicken utricular macula double labeled for the PB473-3 peptide (a, red) and the TLA using mAb G19 (b, green). Image in c is a merge of images shown in a and b. Scale bar, 10 μm. d–f, Double-immunogold labeling for the PB473-3 peptide (5 nm particles; arrowheads) and the TLA (10 nm particles; arrows) in the tip-link region (d, e) and in the stereocilia–kinocilial link region (f) of utricular macular hair cells. S, Stereocilium; K, kinocilium. Scale bars, 100 nm.

Figure 9.

Effects of BAPTA and subtilisin on protocadherin-15 isoform classes. a–l, Mouse organ of Corti, P2 plus 1 d in vitro, double labeled with phalloidin for F-actin (red) and antibodies (green) PB303 to protocadherin-15–CD1 (a–c), PB464-2B to protocadherin-15–CD2 (d–f), PB375 to protocadherin-15–CD3 (g–i), and HL5614 to a recombinant ectodomain fragment of protocadherin-15 (j–l). Cultures were treated with saline (a, d, g, j; Control), 5 mm BAPTA (b, e, h, k; BAPTA), or 50 μg/ml subtilisin (c, f, i, l; Subtilisin) for 15 min at room temperature. Culture in l was exposed to BAPTA after subtilisin treatment to reveal masked epitopes for HL5614. The left half of each panel shows the distribution of protocadherin-15, and the right half shows the merge with F-actin. Scale bar, 10 μm.

Figure 10.

Reappearance of protocadherin-15 epitopes after BAPTA treatment. a–f, P2 mouse organ of Corti, 1 d in vitro, labeled with phalloidin for F-actin (red) and antibody HL5614 to a recombinant ectodomain fragment of protocadherin-15 (a–c, green) and PB375 to protocadherin-15–CD3 (d–f, green). Cultures in a–c were treated as follows at room temperature: a, 5 mm BAPTA for 5 min; b, 5 mm BAPTA for 5 min, followed by saline for 5 min; c, 5 mm BAPTA for 5 min, saline for 5 min, followed by 5 mm BAPTA for 5 min. Cultures in d–f were treated with 5 mm BAPTA for 5 min at room temperature, washed in saline over a 5 min period, and then replaced in culture at 37°C for 1 h (d), 4 h (e), and 24 h (f). The left half of each panel shows the distribution of protocadherin-15, and the right half shows the merge with F-actin. Scale bar, 10 μm.