Visualizing Collagen Fibrils in the Cochlea's Tectorial and Basilar Membranes Using a Fluorescently Labeled Collagen-Binding Protein Fragment.

PURPOSE: A probe that binds to unfixed collagen fibrils was used to image the shapes and fibrous properties of the TM and BM. The probe (CNA35) is derived from the bacterial adhesion protein CNA. We present confocal images of hydrated gerbil TM, BM, and other cochlear structures stained with fluorescently labeled CNA35. A primary purpose of this article is to describe the use of the CNA35 collagen probe in the cochlea. METHODS: Recombinant poly-histidine-tagged CNA35 was expressed in Escherichia coli, purified by cobalt-affinity chromatography, fluorescence labeled, and further purified by gel filtration chromatography. Cochleae from freshly harvested gerbil bullae were irrigated with and then incubated in CNA35 for periods ranging from 2 h - overnight. The cochleae were fixed, decalcified, and dissected. Isolated cochlear turns were imaged by confocal microscopy. RESULTS: The CNA35 probe stained the BM and TM, and volumetric imaging revealed the shape of these structures and the collagen fibrils within them. The limbal zone of the TM stained intensely. In samples from the cochlear base, intense staining was detected on the side of the TM that faces hair cells. In the BM pectinate zone, staining was intense at the upper and lower boundaries. The BM arcuate zone was characterized by a prominent longitudinal collagenous structure. The spiral ligament, limbus and lamina stained for collagen, and within the spiral limbus the habenula perforata were outlined with intense staining. CONCLUSION: The CNA35 probe provides a unique and useful view of collagenous structures in the cochlea.

Structure, Function, and Development of the Tectorial Membrane: An Extracellular Matrix Essential for Hearing.

The tectorial membrane is an extracellular matrix that lies over the apical surface of the auditory epithelia in the inner ears of reptiles, birds, and mammals. Recent studies have shown it is composed of a small set of proteins, some of which are only produced at high levels in the ear and many of which are the products of genes that, when mutated, cause nonsyndromic forms of human hereditary deafness. Quite how the proteins of the tectorial membrane are assembled within the lumen of the inner ear to form a structure that is precisely regulated in its size and physical properties along the length of a tonotopically organized hearing organ is a question that remains to be fully answered. In this brief review we will summarize what is known thus far about the structure, protein composition, and function of the tectorial membrane in birds and mammals, describe how the tectorial membrane develops, and discuss major events that have occurred during the evolution of this extracellular matrix.

Porosity controls spread of excitation in tectorial membrane traveling waves.

Cochlear frequency selectivity plays a key role in our ability to understand speech, and is widely believed to be associated with cochlear amplification. However, genetic studies targeting the tectorial membrane (TM) have demonstrated both sharper and broader tuning with no obvious changes in hair bundle or somatic motility mechanisms. For example, cochlear tuning of Tectb(-/-) mice is significantly sharper than that of Tecta(Y1870C/+) mice, even though TM stiffnesses are similarly reduced relative to wild-type TMs. Here we show that differences in TM viscosity can account for these differences in tuning. In the basal cochlear turn, nanoscale pores of Tecta(Y1870C/+) TMs are significantly larger than those of Tectb(-/-) TMs. The larger pore size reduces shear viscosity (by ∼70%), thereby reducing traveling wave speed and increasing spread of excitation. These results demonstrate the previously unrecognized importance of TM porosity in cochlear and neural tuning.

The 3D structure of the tectorial membrane determined by second-harmonic imaging microscopy.

The tectorial membrane (TM) is a highly hydrated non-cellular matrix situated over the sensory cells of the cochlea. It is widely accepted that the mechanical coupling, between the TM and outer hair cells stereocilia bundles, plays an important role in the cochlea energy transduction mechanism. Recently, we provided supporting evidence for the existence of mechanical coupling by demonstrating that the mechanical properties of the TM change along its longitudinal direction. Since the biochemical composition of the TM is similar throughout its entire length, it is likely that structural differences induce the observed material properties changes. Presently, however, the structure of the TM under physiological environments remains unknown. In this work, the 3D structure of native TM samples is shown by using two-photon second-harmonic imaging microscopy. We find that the collagen fibers at the basal region are arranged in a parallel orientation while being tilted in an angle with respect to the plane of the TM surface at the apical region. Moreover, we find an intensified marginal band at the basal OHC zone which forms a shell-like structure which engulfs the stereocilium imprints surface of the TM. In supports of our previous mechanical characterization, the analysis presented here provides a structural basis for the changes in TM's mechanical properties.

Mice doubly deficient in the midkine and pleiotrophin genes exhibit deficits in the expression of beta-tectorin gene and in auditory response.

alpha-Tectorin and beta-tectorin are major noncollagenous proteins of the tectorial membrane, which plays a crucial role in the reception of sonic signals in the cochlea. Midkine and pleiotrophin are closely related proteins that serve as growth factors and cytokines. In mice doubly deficient in the midkine gene and pleiotrophin gene, expression of beta-tectorin mRNA was nearly abolished in the cochlea on day 1 and 7 after birth. Expression of alpha-tectorin mRNA was unaffected in the double knockout mice, and expression of beta-tectorin mRNA was not altered in mice deficient in only the midkine or pleiotrophin gene. In newborn wild-type mice, both midkine and pleiotrophin were expressed in the greater epithelial ridge of the cochlea, in which beta-tectorin mRNA was strongly expressed. These results indicate that either midkine or pleiotrophin is required for significant expression of beta-tectorin. In agreement with the view that beta-tectorin is essential for normal auditory function, mice doubly deficient in both midkine and pleiotrophin genes exhibited very severe auditory deficits. We observed that mice deficient in either midkine or pleiotrophin gene were also impaired in their auditory response, but the level of the deficit was generally low or moderate. The present finding illustrates the importance of growth factor expression in the cochlea for auditory function.

Collagen changes in the cochlea of aged Fischer 344 rats.

Hearing function in the Fischer 344 (F344) albino inbred strain of rats deteriorates with aging faster than in other strains, in spite of the small hair cell loss in old F344 animals [Popelar, J., Groh, D., Pelanova, J., Canlon, B., Syka, J., 2005. Age-related changes in cochlear and brainstem auditory function. Neurobiol. Aging, in press.]. This study was aimed at elucidating the structural changes in the inner ear of this rat strain during aging. Cochlear histopathology was examined in 20-24-month-old F344 rats and compared with that of young F344 rats (4 months) and of old rats of the Long-Evans (LE) strain. Hematoxylin/eosin staining in aged F344 rats showed degenerative changes in the organ of Corti, consisting of a damaged layer of marginal cells, reduced vascularization of the stria vascularis and a distorted tectorial membrane detached from the organ of Corti. Age-related changes in collagen distribution were observed with Masson's trichrome staining in the spiral ligament of old F344 rats. The results of immunohistochemical staining for type II collagen revealed a marked decrease in collagen fibers in the area connecting the spiral ligament and stria vascularis and a decrease in area IV fibrocytes in old F344 but not in LE rats. These findings may contribute to an explanation of the substantial hearing loss found in old F344 rats.

Sequence similarity between stereocilin and otoancorin points to a unified mechanism for mechanotransduction in the mammalian inner ear.

BACKGROUND: Interaction between hair cells and acellular gels of the mammalian inner ear, the tectorial and otoconial membranes, is crucial for mechanoreception. Recently, otoancorin was suggested to be a mediator of gel attachment to nonsensory cells, but the molecular components of the interface between gels and sensory cells remain to be identified. HYPOTHESIS: We report that the inner ear protein stereocilin is related in sequence to otoancorin and, based on its localisation and predicted GPI-anchoring, may mediate attachment of the tectorial and otoconial membranes to sensory hair bundles. TESTING: It is expected that antibodies directed against stereocilin would specifically label sites of contact between sensory hair cells and tectorial/otoconial membranes of the inner ear. IMPLICATIONS: Our findings support a unified molecular mechanism for mechanotransduction, with stereocilin and otoancorin defining a new protein family responsible for the attachment of acellular gels to both sensory and nonsensory cells of the inner ear.

Extracellular matrices associated with the apical surfaces of sensory epithelia in the inner ear: molecular and structural diversity.

The ultrastructure and molecular composition of the extracellular matrices that are associated with the apical surfaces of the mechanosensory epithelia in the mouse inner ear are compared. A progressive increase in molecular and structural organization is observed, with the cupula being the simplest, the otoconial membrane exhibiting an intermediate degree of complexity, and the tectorial membrane being the most elaborate of the three matrices. These differences may reflect changes that occurred in the acellular membranes of the inner ear as a mammalian hearing organ arose during evolution from a simple equilibrium receptor. A comparison of the molecular composition of the acellular membranes in the chick inner ear suggests the auditory epithelium and the striolar region of the maculae are homologous, indicating the basilar papilla may have evolved from the striolar region of an otolithic organ. A comparison of the tectorial membranes in the chick cochlear duct and the mouse cochlea reveals differences in the structure of the noncollagenous matrix in the two species that may result from differences in the stochiometry of alpha- and beta-tectorin and/or differences in the post-translational modification of alpha-tectorin. This comparison also indicates that the appearance of collagen in the mammalian tectorial membrane may have been a major step in the evolution of an electromechanically tuned vertebrate hearing organ that operates over an extended frequency range.

Equilibrium behavior of an isotropic polyelectrolyte gel model of the tectorial membrane: effect of pH.

An isotropic polyelectrolyte gel model (Weiss and Freeman, 1996a) of the tectorial membrane (TM) was extended to incorporate the effect of pH. The effect of pH is analyzed in order to interpret measurements of the effect of pH on the dimensions of the TM (Freeman et al., 1996a). The pH dependence of the model results from the binding of hydrogen ions to TM macromolecules--to both neutral sites with basic pK and negatively charged sites with acidic pK. Parameters of the model can be based on estimates of the concentration in the TM of collagen and glycosaminoglycans (GAGs), two identified constituents of the TM that account for approximately 40% of its dry weight. The resulting model shows swelling responses at both high and low pH that are qualitatively similar to the measurement on the TM but that differ quantitatively. Alternatively, parameters of the model can be chosen in an ad hoc fashion to closely fit the measurements of TM swelling as a function of pH. Taken together these results suggest that either estimates of the collagen and GAG content of the TM are in error or constituents of the TM, which have not yet been identified, contain appreciable pH-dependent fixed charge and contribute to the swelling behavior of the TM.

Immunohistochemical localization of keratan sulfate in the chinchilla inner ear.

Keratan sulfate (KS) was immunolocalized in the chinchilla cochlea and vestibular system using indirect immunohistochemistry and a monoclonal antibody (clone 5-D-4) directed against a proteoglycan core antigen. As a positive control, anti-KS Mab reactivity was found in the pericellular matrix and lacuna walls of temporal bone osteocytes. In the cochlea, anti-KS Mab reactivity was abundant in the basal cell layer of the stria vascularis and in the marginal band and Hensen's stripe of the tectorial membrane. Less anti-KS Mab reactivity was present in the cover net, Hardesty's membrane and the upper fibrous zone of the limbal layer of the tectorial membrane. In the vestibular system, anti-KS Mab reactivity was immunolocalized to a portion of the epithelium overlying the cupula of the crista ampullaris, in the apical surface of crista ampullaris epithelium, crista ampullaris stereocilia and in the otoconia. Elucidating the distribution of KS in the cochlea will improve our understanding of cochlear anatomy and is a first step toward understanding the etiology of hearing loss observed in diseases involving KS metabolism, namely, mucopolysaccharidosis type IV (Morquio's syndrome).

Immunoreactivity for taurine in the cochlea: its abundance in supporting cells.

Taurine is the second most abundant free amino acid in the brain where its osmoregulatory function is well established. Taurine-deprived kittens show retinal pathology leading to blindness. In the inner ear, taurine has been reported to be the most abundant free amino acid although its role in inner ear function is not known. Immunohistochemistry was employed here to investigate the localisation of taurine in normal cochleae of the guinea pig compared with two different conditions: experimentally induced endolymphatic hydrops and after oral administration of glycerol. In normal cochleae, by light microscopy, taurine-like immunoreaction was never observed in the sensory outer hair cells and appeared absent from the inner hair cells. In contrast taurine-like immunolabeling was found to be present in all supporting tissue with the striking exception of the tectorial membrane and the outer pillar cell which had no or little taurine immunoreactivity respectively. In early experimental endolymphatic hydrops, the distribution of taurine-like immunoreactivity appeared similar to that observed for normal cochleae. In long-term hydrops, degenerated outer hair cells were replaced by the swelling of the phalangeal process of the Deiters' cells which became highly immunoreactive to taurine. After glycerol administration, the tectorial membrane became more tightly bound to the apical surface of the sensory hair cells and distinctly immunoreactive to taurine. The localisation of taurine in the organ of Corti shown here is consistent with taurine being involved in the maintenance of osmotic equilibrium in the normal and perhaps also in the restructuration of the pathological organ of Corti.

The mouse tectorins. Modular matrix proteins of the inner ear homologous to components of the sperm-egg adhesion system.

The cDNA and derived amino acid sequences for the two major non-collagenous proteins of the mouse tectorial membrane, alpha- and beta-tectorin, are presented. The cDNA for alpha-tectorin predicts a protein of 239,034 Da with 33 potential N-glycosylation sites, and that of beta-tectorin a smaller protein of 36,074 Da with 4 consensus N-glycosylation sites. Southern and Northern blot analysis indicate alpha- and beta-tectorin are single copy genes only expressed in the inner ear, and in situ hybridization shows they are expressed by cells both in and surrounding the mechanosensory epithelia. Both sequences terminate with a hydrophobic COOH terminus preceded by a potential endoproteinase cleavage site suggesting the tectorins are synthesized as glycosylphosphatidylinositol-linked, membrane bound precursors, targeted to the apical surface of the inner ear epithelia by the lipid and proteolytically released into the extracellular compartment. The mouse beta-tectorin sequence contains a single zona pellucida domain, whereas alpha-tectorin is composed of three distinct modules: an NH2-terminal region similar to part of the entactin G1 domain, a large central segment with three full and two partial von Willebrand factor type D repeats, and a carboxyl-terminal region which, like beta-tectorin, contains a single zona pellucida domain. The central, high molecular mass region of alpha-tectorin containing the von Willebrand factor type D repeats has homology with zonadhesin, a sperm membrane protein that binds to the zona pellucida. These results indicate the two major non-collagenous proteins of the tectorial membrane are similar to components of the sperm-egg adhesion system, and, as such may interact in the same manner.

Uronic acid-containing glycosaminoglycans and keratan sulfate are present in the tectorial membrane of the inner ear: functional implications.

The tectorial membrane is a gel-like, acellular connective tissue overlying the microscopic organ of Corti--the auditory sensory structure. It is instrumental in the sound-synchronous deflection of the stereocilia of the hair cells, a central event in auditory transduction. It is well established that collagen, primarily type II, constitutes the major protein of the tectorial membrane, with smaller amounts of types IX and XI also present. However, conclusive information on the proteoglycans in this structure is lacking. Tectorial membranes were extracted with a 4 M guanidine--HCl solvent, and proteoglycans isolated after ethanol precipitation and collagenase treatment. A colorimetric assay based on the binding of the cationic dye safranin O to glycosaminoglycans, in combination with enzymatic techniques, detected significant amounts of chondroitin sulfate and keratan sulfate (0.29 and 0.17% on a wet weight basis, respectively). Agarose-polyacrylamide electrophoresis of chondroitinase-digested samples revealed a core protein with a similar molecular mass to that of the large cartilage proteoglycan aggrecan. This proteoglycan reacted with the antibody 3-B-3 (recognizing modified chondroitin 6-sulfate linkage region oligosaccharides). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed several low molecular mass proteins which reacted with 5-D-4, specific for keratan sulfate, one of which showed characteristics of fibromodulin. Comparison of the quantitative aspects of various connective tissue components of tectorial membrane with other type II collagen-containing structures revealed that this tissue resembles highly hydrated cartilage.

Glycoconjugates in the cochlea as revealed by the silver methenamine method.

The glycoconjugates in the cochlea of the guinea pig were studied by staining samples by the silver methenamine method as well as after periodic acid-Schiff (PAS) staining. Results obtained by the two methods were similar but not identical. The silver methenamine method was much better in terms of resolution. However, this method of staining seemed less specific than the PAS reaction. When the silver methenamine method was used, the tectorial membrane and outer hair cells were specifically stained. Two types of fibrils were observed in the tectorial membrane. Thick fibrils were located in the fibrous layer. Thin fibrils were situated in the marginal band, the cover net, Hensen's stripe and the fibrous layer. The thick and thin fibrils appeared to correspond to type A and type B protofibrils, respectively. The outer hair cells were found to contain strongly stained particles which, presumably, consisted of glycogen. The basement membrane of the capillaries in the stria vascularis also gave a positive reaction, while that of other capillaries was essentially unstained. This finding suggests structural differences between these capillaries.

Hearing and glycoconjugates: localization of Le(y), Le(x) and sialosyl-Le(x) in guinea pig cochlea, particularly at the tectorial membrane and sensory epithelia of the organ of Corti.

Immunohistological examination of guinea pig cochleas was performed using a panel of 25 monoclonal antibodies directed to various lacto-, ganglio- and globo-series carbohydrate epitopes as well as mucin-type epitopes. Lacto-series structures were found to be localized at specific sites of the tectorial membrane (TM) and Corti's organ, i.e. alpha 1-->3 fucosyl type 2 chain (Le(x)) at Kimura's membrane, marginal band and covering net of TM; alpha 1-->2, alpha 1-->3 difucosyl type 2 chain (Le(y)) at covering net; and sialosyl-Le(x) and sialosyl-i at Kimura's membrane and sensory epithelia, particularly sensory tips of hair cells of Corti's organ. In striking contrast, ganglio-series structures (GM3, GD3, GD2, 9-O-Ac-GD3) were detected at spiral ganglion cells, neuronal fibres and stria vascularis, but were completely absent from Corti's organ and most of the TM. Other epitope structures defined by various antibodies were not detectable at any location. The functional roles of lacto-series carbohydrate epitopes expressed at TM and Corti's organ remain unknown. However, the expression of Le(y) (but not other structures) in association with developmental deficiency of TM induced by 6-N-propyl-2-thiouracil in rats suggests that Le(y) plays some role in normal TM development. The presence of Le(x) at Kimura's membrane and sialosyl-Le(x) at hair cell sensory tips of Corti's organ suggests the intriguing possibility that these fucosylated/sialosylated carbohydrate structures play some role in interactions (either attractive or repulsive) of these inner ear components, which have been implicated in the physiology of hearing, i.e. the conversion of sound waves to nerve impulses.

The protein composition of the avian tectorial membrane.

Gel electrophoretic analysis of the avian tectorial membrane under non-reducing conditions reveals the presence of 2 major proteins with apparent molecular masses of 195 and 41 kDa on 8.25% gels. Under reducing conditions, 6 polypeptides with apparent molecular masses of 146, 60, 56, 43, 35 and 31 kDa are consistently observed. None of these six polypeptides observed under reducing conditions are sensitive to digestion with collagenase, and all, except for the 43 kDa component, are degraded by treatment with cold acidic pepsin. The 60, 56 and 43 kDa polypeptides bind the peroxidase conjugated lectins from Canavalia ensiformis and Triticum vulgaris, indicating the presence of mannose, N-acetyl glucosamine and/or sialic acid. The 146, 60 and 56 kDa bands undergo a shift in electrophoretic mobility after treatment of native tectorial membranes with the enzyme neuroaminidase. Fibronectin and Type II collagen cannot be detected in the avian tectorial membrane by either immunoblotting or immunofluorescence techniques. Polyclonal antisera raised against the different polypeptides after partial purification by one dimensional gel electrophoresis confirm that these proteins are all components of the tectorial membrane, and show that they are restricted to the otolithic and tectorial membranes within the inner ear. Analysis of a wide variety of other tissue types indicates that the 60, 43 and 35 kDa components can only be detected within the inner ear, and that the antisera recognising the 146 and 31 kDa components only show cross-reactivity within the head, with the anti-146 kDa antibodies staining the mucus ducts supplying the olfactory epithelium and the anti-31 kDa antibodies staining granular elements in the cells of the respiratory epithelium. The results suggest that certain of the tectorial membrane components may be novel matrix molecules unique to the inner ear, and that some of the other proteins may be antigenically related to mucins.

Quantitative carbohydrate analyses of the tectorial and otoconial membranes of the guinea pig.

Carbohydrate composition of the tectorial membrane (TM) and the otoconial membrane (OM) of the guinea pig was analyzed after hydrolysis, using high-performance anion-exchange chromatography and pulsed amperometric detection. Both of the tissues were highly glycosylated; the carbohydrate content being 24-42% of protein. GlcN, Gal, Glc and Man were found to be the major component sugars of TM, whereas little GalN was found. Fuc and NANA were also present, but NGNA was not detectable. After digestion with thermolysin for solubilization, OM was separated into two fractions: insoluble mineral particles of the otoconia (OM-ppt) and a soluble fraction from the gelatinous layer (OM-sup). These two fractions showed distinct carbohydrate composition from each other. Further analyses using glycosidases revealed that TM contained asialyl and monosialyl but little di-, tri- and tetrasialyl N-glycosides, and OM-sup did not seem to be susceptible to endo-beta-galactosidase, which is known to cleave some N-acetyl-polylactosamine and keratan sulfate. Based on these analyses, it can be suggested that most of the carbohydrates in TM are likely to be asialyl and monosialyl N-glycosides. N-Glycosides may be predominant in the otoconia as well, and a polymer structure consisting of GlcN(Ac) and Gal other than N-acetyl-polylactosamine may exist in the gelatinous layer of OM. O-Glycosylation of the usual type appeared to be minor in all the fractions.

Type II and type IX collagen form heterotypic fibers in the tectorial membrane of the inner ear.

The presence of type II and IX collagen in the adult gerbil inner ear was probed by use of preembedding and post-embedding immunocytochemistry. Monoclonal antibodies to type II and IX collagen both label the tectorial membrane, an acellular structure which lies over the cochlear sensory hair cells and plays an essential role in the transduction process. At the light microscopic level, the antibodies are localized throughout the tectorial membrane. At the electron microscopic level, antibodies against types II and IX collagen are co-localized over the thick unbranched (Type A) radial fibers but not over the thin highly branched (Type B) fibers in which the thick fibers are embedded. Thus, the tectorial membrane of the cochlea represents another non-cartilaginous structure in which type II and IX collagen are present and arranged in heterotypic fibers. The organization of these fibers into bundles, meshworks and layers results in the formation of a structure with the unique properties necessary to withstand mechanical stresses associated with sensory transduction.