Abstract
Immunological techniques have been used to generate both polyclonal and monoclonal antibodies specific for the apical ends of sensory hair cells in the avian inner ear. The hair cell antigen recognized by these antibodies is soluble in nonionic detergent, behaves on sucrose gradients primarily as a 16S particle, and, after immunoprecipitation, migrates as a polypep-tide with a relative molecular mass Of 275 kD on 5 % SDS gels under reducing conditions. The antigen can be detected with scanning immunoelectron microscopy on the apical surface of the cell and on the stereocilia bundle but not on the kinocilium. Double label studies indicate that the entire stereocilia bundle is stained in the lagena macula (a vestibular organ), whereas in the basilar papilla (an auditory organ) only the proximal region of the stereocilia bundle nearest to the apical surface is stained. The monoclonal anti-hair cell anti-bodies do not stain brain, tongue, lung, liver, heart, crop, gizzard, small intestine, skeletal muscle, feather, skin, or eye tissues but do specificall3, stain renal corpuscles in the kidney. Experiments using organotypic cultures of the embryonic lagena macula indicate that the antibodies cause a significant increase in the steady-state stiffness of the stereocilia bundle but do not inhibit mechanotransduction. The antibodies should provide a suitable marker and/or tool for the purification of the apical sensory membrane of the hair cell. T HE mechanosensory hair cells of the acousticolateralis sense organs of vertebrates are epithelial cells capable of responding to displacements of less than a few nano-meters. Hair cells are both morphologically and functionally polarized, with an apical end specialized for the detection of stimuli and a basolateral surface specialized for the release of neurotransmitter. A highly organized bundle of stereocilia is located at the apical end of the hair cell, and displacement of this stereocilia bundle results in the gating of nonspecific cation channels located in the apical membrane. Depolariza-tion of the hair cell resulting from channel opening causes neurotransmitter to be released around the basolateral surface , and information about mechanical events at the periph-ely is thereby transmitted to contacting afferent nerve fibers and relayed to the central nervous system. Although much is known about both the physiology and morphology of hair cells and the biophysics of mechanotransduction, the molecular properties of the apical surface membrane of the hair cell remain completely uncharacterized. To understand more about the molecular events underlying mechanotransduction, the proteins involved in this process will have to be identified. Purification and characterization of the apical plasma membrane of the hair cell will, however, be difficult since no specific markers or ligands are known for this surface of the cell. We have, therefore, used an im-munological approach to generate such a ligand. This study describes the production of antibodies that are specific, within the inner ear, for the apical end of the hair cell and the identification of a 275-kD protein that is associated with the sensory membrane. Materials and Methods Immunization Lagenae maculae were obtained from inner ears of l-3-d-old domestic fowl, and a crude membrane fraction was prepared by differential centrifugation. Anti-lagena macula sera were produced by immunizing BALB/c mice (Charles River Breeding Laboratories, Ltd., Manston, UK) intraperitone-ally three times at l-too intervals. Material from 200 ears was used for each injection. Animals were test bled at times ranging from 1 to 12 mo after the last injection. Absorbing Polyclonal Sera Anti-lagena macula sera were absorbed sequentially with (a) homogenates prepared from cochlear tissues that do not contain hair cells (i.e., the teg-mentum vasculosum and the cochlear ganglion; see Fig. 1); (b) a homoge-nate from the small intestine; and (c) a mixture of rectorial membranes and otoliths. Tectorial membranes and otoliths were extracted twice with 1% (vol/vol) Triton X-100 (TX-100) l in PBS before use as absorbents. Amounts of tissue required were determined empirically for each test bleed, and the efficacy of the absorption procedure was monitored by immunofluorescence microscopy. Immune sera from two different mice (ISI and IS2) were used in this study. L Abbreviations used in this paper: TBS/HS, TBS containing 10% heat-inactivated horse serum; TX-100, Triton X-100.