The Journal of Neuroscience, June 28, 2006, ():
The Tip-Link Antigen, a Protein Associated with the Transduction Complex of Sensory Hair Cells, Is Protocadherin-15
J. Neurosci. Ahmed et al.
26: 7022
Supplemental data
Files in this Data Supplement:
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Table S1. PCR primers
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Table S2. Antigens used to raise antisera to protocadherin-15
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Table S3. Accession numbers for protocadherin-15 isoforms
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Figure S1. (a-c) The amino acid sequences encoded by exons 35, 38 and 39 of mouse protocadherin-15. In panels a-c, each of the three cytoplasmic domains has a unique class I PDZ ligand (underlined) on the carboxy-terminus. Proline residues are highlighted in purple, serine residues in pink and glutamic acid residues in green. (a) Exon 35 encoding the unique part of protocadherin-15-CD1 has proline rich regions (highlighted in purple). (b) Exon 38 encoding the unique part of protocadherin-15-CD2 has a cytoplasmic domain with stretches of glutamic acid residues (highlighted in green). (c) Exon 39 encoding the unique part of protocadherin-15-CD3.
(d) Composite cytoplasmic domains of protocadherin-15-CD1, protocadherin-15-CD2 and protocadherin-15-CD3. cDNAs from mouse inner ear and human retina were PCR amplified using primers in exon 31 and 39 of Pcdh15/PCDH15. Isoform c1 has 88 amino acid residues encoded by part of exon 35, exons 36 and 37, which are spliced in frame to exon 39. Isoform c2 has 88 amino acid residues encoded by exon 35, exons 36 and 37, and 257 residues encoded by exon 38, which are spliced in frame to exon 39.
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Figure S2. Reactivity of antibodies to protocadherin-15 in av-3J mouse cochlea.
Confocal images of hair bundles in the early postnatal (P3) cochlea of heterozygous (a, c, e, g, i) and homozygous (b, d, f, h, j) av-3J mice stained with antibody PB303 to protocadherin-15-CD1 (a, b), antibody PB464-2B to protocadherin-15-CD2 (c, d), antibody PB375 to protocadherin-15-CD3 (e, f), antibody PB473-3 to an N-terminal peptide in the ectodomain of protocadherin-15 and (f, g) antibody HL5614 to an expression fusion protein corresponding to residues 28-335, which includes EC1 and EC2 (Figure 1) of the extracellular domain of protocadherin-15. The left half of each panel shows the distribution of protocadherin-15, the right half shows the merge with F-actin. Scale bar, 10 μm.
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Figure S3. Specificity of protocadherin-15 antibodies. Confocal images of lymphoblastoid (a, b) and HeLA (c-l) cells that were transfected with a His-tagged Pcdh15-CD1 construct (a, b, i-l), a Flag-tagged CD2 cytoplasmic domain construct (c, d) and a Flag-tagged CD3 cytoplasmic domain construct (e-h) and then double labelled with anti-protocadherin-15 antibodies (green) that had been pre-absorbed with their respective antigens as a positive control (left side, relevant antigen, a, c, e, g, i, k) or with an irrelevant peptide as a negative control (right side, irrelevant antigen, b, d, f, h, j, l) and antibodies to the epitope tags for each construct (red). For each condition the left panel shows a Nomarski image of the cells, the center panel (green) shows the staining seen with the antibodies to protocadherin-15, and the right panel shows the merge with the tagged-construct. Antibodies used are (a) PB303 absorbed with CD1 peptide, (b) PB303 absorbed with a mixture of CD2 and CD3 peptides, (c) PB464-2B absorbed with CD2 peptide, (d) PB464-2B absorbed with a mixture of CD1 and CD3 peptides, (e) PB375 absorbed with CD3 peptide, (f) PB375 absorbed with a mixture of CD1 and CD2 peptides, (g) HL5383 absorbed with expressed CD3 fragment, (h) HL5383 absorbed with CD3 peptide, (i) HL5614 absorbed with expressed ectodomain fragment, (j) HL5614 absorbed with CD1 peptide, (k) PB473-3 absorbed with N-terminal peptide antigen, (l) PB473-3 absorbed with CD1 peptide. Constructs used for transfection are indicated to the left, antisera used are indicated to the right. Scale bars, 5 μm.
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Figure S4. Distribution of protocadherin-15-CD2 in the developing hair bundle.
Confocal images of hair bundles double-labelled with antibody PB464-2B against protocadherin-15-CD2 (green) and phalloidin to detect F-actin (red). The distribution of protocadherin-15-CD2 (left panel), F-actin (middle panel) and the merge of the two labels (right panel) is shown for the rat utricle at E18.5 (a) and the rat ampulla at P7 (b). Protocadherin-15-CD2 is detected in stereocilia of immature hair bundles (asterisks), and in the kinocilia (arrows) of the mature hair bundles. Newly forming hair cells have stereocilia bundles that stain brightly with antisera against CD2 but only stain weakly with phalloidin, presumably due to the low amount of filamentous actin present in immature hair bundles as previously reported (Bartolami et al., 1991; Kondo et al. 2002). Scale bar, 10 μm.
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Figure S5. Summary of the locations of protocadherin-15-CD1, protocadherin-15-CD2 and protocadherin-15-CD3 in developing and mature hair bundles
Schematic diagrams of (a) the mature organ of Corti (b), an immature cochlear hair bundle at P2, and (c) a mature hair bundle at P19. At P2 (b) the links present include the tectorial membrane attachment crowns, tip links, transient lateral links, ankle links, and kinocilial links. In the mature hair bundle at P19 (c) the ankle and kinocilial links have disappeared, and horizontal top connectors have become prominent. The locations of protocadherin-15-CD1, protocadherin-15-CD2 and protocadherin-15-CD3 are shown as green, blue and pink dots, respectively. Protocadherin-15-CD2 is expressed transiently in the hair bundle, may be associated with the kinocilial links and transient lateral links, and could work in tandem with cadherin 23 to maintain the structural integrity of developing hair bundles. Protocadherin-15-CD3 on the top of the shorter stereocilia may directly interact homophilically with protocadherin-15-CD1 on the side of the adjacent taller stereocilia, or indirectly interact through an adaptor-linker protein. This would allow tip links to form during development of stereocilia and regenerate in the mature inner ear after breakage. The inset in panel c depicts protocadherin-15-CD3 contributing to the tip link anchoring elements and/or the tip-link filament.
