Radixin is a constituent of stereocilia in hair cells F. Pataky, R. Pironkova, and A. J. Hudspeth* Howard Hughes Medical Institute and Laboratory of Sensory Neuroscience, The Rockefeller University, 1230 York Avenue, New York, NY 10021 Contributed by A. J. Hudspeth, December 26, 2003 Proteins of the ezrin-radixin-moesin family are ubiquitous constit- present study whether hair cells express an ERM protein that uents of the submembrane cortex, especially in epithelial cells. occurs in hair bundles. Earlier biochemical results suggested that a protein of this family occurs in the hair bundle, the cluster of actin-filled stereocilia that Materials and Methods serves as the mechanoreceptive organelle of each hair cell in the Production of Antisera. For the production of antisera, we selected inner ear. We prepared antipeptide antisera directed against portions of chicken ezrin and radixin corresponding to regions in chicken radixin and ezrin and demonstrated their specificity and the human proteins that had previously proven useful in eliciting absence of crossreactivity. When used in immunocytochemical specific immune responses (9). Antisera 1041 and 1029 were studies of isolated hair cells, anti-radixin produced an intense band raised against two distinct portions of chicken radixin (GenBank of labeling at the bases of hair bundles from the chicken, frog, accession no. CAB59977), NH2-478-VIPPTENEHDEHDENN- mouse, and zebrafish. Electron microscopic immunocytochemistry COOH and NH2-400-KAALAKQAADQMKN-COOH, respec- disclosed that radixin labeling commenced in the stereociliary tively. These sequences are specific for radixin, with little taper, peaked in the lower stereociliary shaft, and declined pro- homology to avian ezrin or to the moesins of other species. gressively toward the hair bundle’s top. Labeling with anti-ezrin Antiserum 1028 was raised against a peptide from chicken ezrin produced no signal in hair bundles. Radixin is thus a prominent (GenBank accession no. BAA75497) NH2-476-IYEPVNYH- constituent of stereocilia, where it may participate in anchoring the VHDNLHDEGSEY-COOH, with minimal homology to avian ‘‘pointed’’ ends of actin filaments to the membrane. radixin or to moesins. The synthesis of each peptide, conjugation to keyhole limpet hemocyanin by an amino-terminal cysteine he hair bundle is the mechanoreceptive organelle essential residue, purification, and serum production in rabbits were Tfor the senses of hearing and equilibrium throughout the performed commercially (Covance Research Products). To NEUROSCIENCE vertebrates. Projecting from the apical surface of a hair cell, the demonstrate immunoreactivity, preliminary and production bundle comprises some 20–300 rigid, cylindrical stereocilia from bleeds were tested against preimmune sera by immunoblotting. Ϸ1toϷ50 ␮m in length. The stereocilia grow successively longer Preimmune sera displayed only weak, nonspecific binding to along one axis of the hexagonal array in which they are packed, isolated brain and cochlear proteins, whereas antisera 1041, so that a bundle displays a beveled top (1). In the developing ear, 1028, and 1029 showed little background but strong reactions a single kinocilium endowed with an axoneme marks the center with ERM proteins. Although both antisera against chicken of the bundle’s tall edge. This organelle is not essential for radixin produced similar results in immunoblotting and immu- nocytochemical labeling, antiserum 1041 generally yielded stron- mechanoelectrical transduction, however, for it degenerates in ger signals. This antiserum was therefore used in all of the the mature mammalian cochlea and can be dissected away immunocytochemical experiments reported here, save for label- without an obvious effect on mechanosensitivity (2). ing of zebrafish hair bundles, for which antiserum 1029 proved The normal hair bundle plays an essential role in the process superior. All antisera were used without further purification. of mechanoelectrical transduction (3). Moreover, many forms of genetic, traumatic, pharmacological, and geriatric deafness and Immunoblotting. Precast 4–20% Tris-glycine gels were used for dysequilibrium stem from bundle degeneration. Identification of the initial characterization of sera. Peptide competition and the hair bundle’s biochemical constituents is therefore essential immunoprecipitation experiments involved 6% Tris-glycine gels. for a complete understanding of normal and pathological hear- Electrophoresis was conducted at 4°C for2hat125Vwith ing and balance. Although a hair bundle contains hundreds of Tris͞glycine͞SDS running buffer containing 1 mM EDTA. proteins (4), only 20 or so have been identified. Biochemical, Proteins were transferred at 4°C for 80 min at 85 V to nitrocel- immunocytochemical, genetic, and physiological techniques are lulose membranes, which were washed in cold PBS containing therefore being applied intensively to recognize the components 0.1% Tween 20, the medium used in all subsequent steps. After essential to mechanoelectrical transduction. having been blocked with 5% nonfat dry milk, membranes were In the course of analyzing the proteins isolated from hair incubated for3hatroom temperature with the appropriate bundles of the bullfrog’s sacculus by PAGE, we noted one with antisera. They were then washed and exposed for 1 h to an unusual feature (4). A molecule of an apparent molecular Ϸ horseradish peroxidase-conjugated donkey anti-rabbit immuno- mass of 77 kDa seemed to occur at only a modest concentration globin (Amersham Pharmacia Biosciences) at a dilution of when assayed by silver staining. When bundle components were 1:10,000. Finally, the membranes were washed, developed, and instead detected after labeling with N-hydroxysulfosuccinim- exposed. idobiotin; however, the protein became prominent. Because N-hydroxysuccinimides attack amino groups, this behavior sug- Peptide Competition Experiments. To prepare samples for immu- gested that the protein is especially rich in lysine residues. The noblotting, brains were dissected from chickens 1–2 weeks of age labeling pattern of the substance indicated that it is an intracel- and Ϸ1 g of tissue was treated with 10 ml of lysis solution lular protein poorly extracted by nonionic detergent (4), as might containing 150 mM NaCl, 1% Triton X-100, and 40 mM Tris, pH be expected for a component of the cytoskeleton or the sub- 7.4, with a protease inhibitor mixture (EDTA-free Complete, membrane cortex. Although these features are scarcely diagnos- tic, they are characteristic of members of the ezrin-radixin- moesin (ERM) protein family (5–8). Moreover, proteins of this Abbreviation: ERM, ezrin-radixin-moesin. family are often constituents of epithelial cells, and especially of *To whom correspondence should be addressed at: The Rockefeller University, Box 314, their apical protrusions such as the microvilli from which 1230 York Avenue, New York, NY 10021-6399. E-mail: [email protected]. stereocilia originate (1). We have therefore inquired in the © 2004 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0308620100 PNAS ͉ February 24, 2004 ͉ vol. 101 ͉ no. 8 ͉ 2601–2606 Downloaded by guest on October 2, 2021 Roche). Thirty chicken cochleae were treated with 200 ␮l of lysis incubation with primary antiserum, all steps were conducted at solution. Brush borders for protein extraction were obtained by room temperature. After a 30-min wash in PBS, cells were following a published protocol (10) that was simplified by observed in PBS with a laser-scanning confocal microscope. omission of the sucrose gradient and use of intestinal villi as a For labeling of filamentous actin, Alexa Fluor 568-phalloidin crude sample. Approximately 1 ml of sedimented intestinal villi (Molecular Probes) was added at a concentration of 50 ng͞ml was resuspended in 10 ml of lysis solution. Solubilization was during the final 30 min of the incubation with secondary performed by dispersing each sample with a glass homogenizer antibodies. For staining of nuclei in preparations of intestinal and incubating for 30 min at 4°C. Each sample was then epithelial cells, TO-PRO-3 iodide (Molecular Probes) was in- centrifuged for 10 min at 4°C at 12,000 ϫ g and the supernatant cluded at a concentration of 1 ␮g͞ml throughout the secondary was retained. After protein concentrations had been deter- antibody incubation. mined, 25 ␮g of brush-border, brain, or cochlear protein was loaded per lane on two identical gels for each antiserum to be Electron Microscopic Immunocytochemistry. Cochleae from chick- tested. After electrophoresis and transfer had been conducted as ens Ϸ3 weeks old were dissected in artificial avian perilymph described above, membranes were blocked overnight. For each consisting of 145 mM Naϩ,2mMKϩ,2mMCa2ϩ,2mMMg2ϩ, Ϫ Ϫ pair of identical membranes, two solutions were prepared: one 120 mM Cl ,22mMD-glucuronate, 11 mM HCO3 , 0.1 mM 2Ϫ containing antiserum at a dilution of 1:2,500 in PBS containing HPO4 , 5 mM sucrose, 5 mM D-glucose, 1 mM sodium pyru- 5% nonfat dry milk and 0.1% Tween 20, the other with the same vate, 1 mM creatine, and 5 mM Hepes, pH 7.4. After removal of constituents plus 100 ␮g͞ml peptide 1041, 1028, or 1029. Solu- tions were incubated at room temperature for 1 h before application to membranes and incubation for3hatroom temperature with mild agitation. The immunoblots were then washed and developed as described above. Immunoprecipitation and Immunoblotting. We immunoprecipi- tated proteins from the brain or