Repair of hair cells following mild trauma may involve extracellular chaperones

Sea anemones were subjected to mild trauma consisting of a 2 min immersion in calcium-depleted seawater. The trauma caused a loss of vibration sensitivity that spontaneously recovered within 50 min of returning the anemones to calcium containing seawater. Apparently, recovery is conferred by proteins contained in fraction gamma, a chromatographic fraction of homogenized mucus collected at the base of anemones allowed to recover from similar trauma. On silver stained SDS-PAGE gels, fraction gamma consists of a single band having an estimated mass of 55 kDa. Fraction gamma is alone sufficient to repair hair bundle mechanoreceptors in anemones. Its biological activity is enhanced in the presence of exogenously supplied ATP, but not GTP nor ADP-ribose. Biotinylated fraction gamma binds to hair bundles. The hypothesis that fraction gamma consists of Hsp60 proteins was tested. Commercial antibodies to Hsp60 label a band at 55 kDa in western blots. Hsp60 antibodies label hair bundles in traumatized anemones but not in untreated controls. Dilute Hsp60 antiserum (but not nonimmune serum) delays the spontaneous recovery of vibration sensitivity in anemones subjected to mild trauma. Thus, fraction gamma likely consists of Hsp60, or a Hsp60-like protein, that functions on the extracellular face of the plasma membrane to restore function to traumatized hair bundles.     

Reorganization of actin during repair of hair bundle mechanoreceptors

Hair bundle mechanoreceptors can be damaged by over-stimulation or by exposure to calcium-free buffers. Provided the trauma is slight, hair bundles recover, although the subcellular mechanisms for such recovery are poorly understood. Hair bundle mechanoreceptors on tentacles of sea anemones are especially resilient, recovering from severe trauma within several hours. During the recovery period, large protein complexes are secreted called “repair proteins” containing replacement linkages for those lost during trauma. In the present study, we find that recovery requires reorganization of the actin-based cytoskeleton in hair bundles. F-actin is first partially depolymerized and then repolymerized in hair bundles based on confocal microscopy. Furthermore, stereocilia show considerable motility during repair based on field emission scanning electron microscopy of hair bundles fixed at 1 min intervals after exposure to exogenously supplied repair protein complexes. Recovery of vibration sensitivity occurs at the organismal level within 8 min. Paradoxically, a full recovery of morphology of hair bundles requires approximately 45 min and a recovery of F-actin levels requires approximately 40 min. Similarly, a full recovery of mechanoelectric responses of hair cells requires approximately 45 min. Thus, it appears that the recovery of responsiveness at the organismal level precedes a full recovery of hair bundles.     

Cadherin 23 is a component of the transient lateral links in the developing hair bundles of cochlear sensory cells

Cadherin 23 is required for normal development of the sensory hair bundle, and recent evidence suggests it is a component of the tip links, filamentous structures thought to gate the hair cells' mechano-electrical transducer channels. Antibodies against unique peptide epitopes were used to study the properties of cadherin 23 and its spatio-temporal expression patterns in developing cochlear hair cells. In the rat, intra- and extracellular domain epitopes are readily detected in the developing hair bundle between E18 and P5, and become progressively restricted to the distal tip of the hair bundle. From P13 onwards, these epitopes are no longer detected in hair bundles, but immunoreactivity is observed in the apical, vesicle-rich, pericuticular region of the hair cell. In the P2-P3 mouse cochlea, immunogold labeling reveals cadherin 23 is associated with kinocilial links and transient lateral links located between and within stereociliary rows. At this stage, the cadherin 23 ectodomain epitope remains on the hair bundle following BAPTA or La(3+) treatment, but is lost following exposure to the protease subtilisin. In contrast, mechano-electrical transduction is abolished by BAPTA but unaffected by subtilisin. These results suggest cadherin 23 is associated with transient lateral links that have properties distinct from those of the tip-link.     

A core cochlear phenotype in USH1 mouse mutants implicates fibrous links of the hair bundle in its cohesion, orientation and differential growth

The planar polarity and staircase-like pattern of the hair bundle are essential to the mechanoelectrical transduction function of inner ear sensory cells. Mutations in genes encoding myosin VIIa, harmonin, cadherin 23, protocadherin 15 or sans cause Usher syndrome type I (USH1, characterized by congenital deafness, vestibular dysfunction and retinitis pigmentosa leading to blindness) in humans and hair bundle disorganization in mice. Whether the USH1 proteins are involved in common hair bundle morphogenetic processes is unknown. Here, we show that mouse models for the five USH1 genetic forms share hair bundle morphological defects. Hair bundle fragmentation and misorientation (25-52 degrees mean kinociliary deviation, depending on the mutant) were detected as early as embryonic day 17. Abnormal differential elongation of stereocilia rows occurred in the first postnatal days. In the emerging hair bundles, myosin VIIa, the actin-binding submembrane protein harmonin-b, and the interstereocilia-kinocilium lateral link components cadherin 23 and protocadherin 15, all concentrated at stereocilia tips, in accordance with their known in vitro interactions. Soon after birth, harmonin-b switched from the tip of the stereocilia to the upper end of the tip link, which also comprises cadherin 23 and protocadherin 15. This positional change did not occur in mice deficient for cadherin 23 or protocadherin 15. We suggest that tension forces applied to the early lateral links and to the tip link, both of which can be anchored to actin filaments via harmonin-b, play a key role in hair bundle cohesion and proper orientation for the former, and in stereociliary elongation for the latter.     

Spatiotemporal pattern and isoforms of cadherin 23 in wild type and waltzer mice during inner ear hair cell development

Mutant alleles of the gene encoding cadherin 23 are associated with Usher syndrome type 1 (USH1D), isolated deafness (DFNB12) in humans, and deafness and circling behavior in waltzer (v) mice. Stereocilia of waltzer mice are disorganized and the kinocilia misplaced, indicating the importance of cadherin 23 for hair bundle development. Cadherin 23 was localized to developing stereocilia and proposed as a component of the tip link. We show that, during development of the inner ear, cadherin 23 is initially detected in centrosomes at E14.5, then along the length of emerging stereocilia, and later becomes concentrated at and subsequently disappears from the tops of stereocilia. In mature vestibular hair bundles, cadherin 23 is present along the kinocilium and in the region of stereocilia-kinocilium bonds, a pattern conserved in mammals, chicks, and frogs. Cadherin 23 is also present in Reissner's membrane (RM) throughout development. In homozygous v(6J) mice, a reported null allele, cadherin 23 was absent from stereocilia, but present in kinocilia, RM, and centrosomes. We reconciled these results by identifying two novel isoforms of Cdh23 unaffected in sequence and expression by the v(6J) allele. Our results suggest that Cdh23 participation in stereocilia links may be restricted to developing hair bundles.     

Evidence for involvement of TRPA1 in the detection of vibrations by hair bundle mechanoreceptors in sea anemones

A homolog of TRPA1 was identified in the genome of the anemone, Nematostella vectensis (nv-TRPA1a), and predicted to possess six ankyrin repeat domains at the N-terminus and an ion channel domain near the C-terminus. Transmembrane segments of the ion channel domain are well conserved among several known TRPA1 polypeptides. Inhibitors of TRPA1 including ruthenium red decrease vibration-dependent discharge of nematocysts in N. vectensis and Haliplanella luciae. Activators of TRPA1 including URB-597 and polygodial increase nematocyst discharge in the absence of vibrations. Co-immunoprecipitation yields a band on SDS-PAGE gels at the predicted mass of the nv-TRPA1a polypeptide among other bands. Co-immunoprecipitation performed in the presence of antigenic peptide decreases the yield of this and several other polypeptides. In untreated controls, anti-nv-TRPA1a primarily labels the base of the hair bundle with some labeling also distributed along the length of stereocilia. Tissue immunolabeled in the presence of the antigenic peptide exhibits reduced labeling. Activating chemoreceptors for N-acetylated sugars induce immunolabel to distribute distally in stereocilia. In anemones, activating chemoreceptors for N-acetylated sugars induce hair bundles to elongate among several other structural and functional changes. Taken together, these results are consistent with the possibility that nv-TRPA1a participates in signal transduction of anemone hair bundles.     

Myosin VIIa,harmonin and cadherin 23, three Usher I gene products that cooperate to shape the sensory hair cell bundle

Deaf-blindness in three distinct genetic forms of Usher type I syndrome (USH1) is caused by defects in myosin VIIa, harmonin and cadherin 23. Despite being critical for hearing, the functions of these proteins in the inner ear remain elusive. Here we show that harmonin, a PDZ domain-containing protein, and cadherin 23 are both present in the growing stereocilia and that they bind to each other. Moreover, we demonstrate that harmonin b is an F-actin-bundling protein, which is thus likely to anchor cadherin 23 to the stereocilia microfilaments, thereby identifying a novel anchorage mode of the cadherins to the actin cytoskeleton. Moreover, harmonin b interacts directly with myosin VIIa, and is absent from the disorganized hair bundles of myosin VIIa mutant mice, suggesting that myosin VIIa conveys harmonin b along the actin core of the developing stereocilia. We propose that the shaping of the hair bundle relies on a functional unit composed of myosin VIIa, harmonin b and cadherin 23 that is essential to ensure the cohesion of the stereocilia.     

Dynamic tuning of hair bundle mechanoreceptors in a sea anemone during predation

The sea anemone Haliplanella luciae (Cnidaria, Anthozoa) detects chemical and mechanical stimuli from prey. Hair bundle mechanoreceptors on the tentacles participate in regulating discharge of microbasic p-mastigophore nematocysts. Properly stimulated hair bundles sensitize the anemone to discharge nematocysts into objects that contact the tentacles. The hair bundle mechanoreceptors are composed of stereocilia derived from a multicellular complex. This complex consists of a single sensory neuron surrounded by two to four supporting cells. The mechanoreceptor is similar in many ways to vertebrate hair cells of the acousticolateralis system. However, anemone hair bundles are adjustable in structure and responsiveness according to the activity of two different chemoreceptors. One chemoreceptor binds N -acetylated sugars and the other binds amino compounds including proline. N -acetylated sugars induce lengthening of the hair bundle and a downward shift in frequencies that elicit maximal discharge of microbasic p-mastigophore nematocysts. Furthermore, N -acetylated sugars shift maximal discharge to smaller amplitude vibrations. Thus, N -acetylated sugars likely tune hair bundles so that small, swimming zooplankton stimulate maximal discharge. Proline leaks into the seawater from the hemolymph of wounded prey. Proline induces shortening of the hair bundle and shifts maximal discharge of nematocysts to higher frequencies and to larger amplitude vibrations. Thus, proline likely tunes hair bundles so that small, wounded, prey stimulate maximal discharge of nematocysts as they struggle to escape. Thus, suitably sized prey stimulate maximal discharge of microbasic p-mastigophore nematocysts upon first contacting the anemone tentacle and again upon attempting to escape.     

Cadherins and mechanotransduction by hair cells

Mechanotransduction, the conversion of a mechanical stimulus into an electrical signal is crucial for our ability to hear and to maintain balance. Recent findings indicate that two members of the cadherin superfamily are components of the mechanotransduction machinery in sensory hair cells of the vertebrate inner ear. These studies show that cadherin 23 (CDH23) and protocadherin 15 (PCDH15) form several of the extracellular filaments that connect the stereocilia and kinocilium of a hair cell into a bundle. One of these filaments is the tip link that has been proposed to gate the mechanotransduction channel in hair cells. The extracellular domains of CDH23 and PCDH15 differ in their structure from classical cadherins and their cytoplasmic domains bind to distinct effectors, suggesting that evolutionary pressures have shaped the two cadherins for their function in mechanotransduction.     

Hair cells, plasma membrane Ca²⁺ ATPase and deafness

Hearing relies on the ability of the inner ear to convert sound waves into electrical signals. The main actors in this process are hair cells. Their stereocilia contain a number of specific proteins and a scaffold of actin molecules. They are organized in bundles by tip-link filaments composed of cadherin 23 and protocadherin 15. The bundle is deflected by sound waves leading to the opening of mechano-transduction channels and to the influx of K(+) and Ca(2+) into the stereocilia. Cadherin 23 and the plasma membrane calcium ATPase isoform 2 (PMCA2) are defective in human and murine cases of deafness. While the involvement of cadherin 23 in deafness/hearing could be expected due to its structural role in the tip-links, that of PMCA2 has been discovered only recently. This review will summarize the structural and functional characteristics of hair cells, focusing on the proteins whose mutations may lead to a deafness phenotype.     

In search of the hair-cell gating spring elastic properties of ankyrin and cadherin repeats

Mechanotransduction in vertebrate hair cells involves a biophysically defined elastic element (the "gating spring") that pulls on the transduction channels. The tip link, a fine filament made of cadherin 23 linking adjacent stereocilia in hair-cell bundles, has been suggested to be the gating spring. However, TRP channels that mediate mechanotransduction in Drosophila, zebrafish, and mice often have cytoplasmic domains containing a large number of ankyrin repeats that are also candidates for the gating spring. We have explored the elastic properties of cadherin and ankyrin repeats through molecular dynamics simulations using crystallographic structures of proteins with one cadherin repeat or 4 and 12 ankyrin repeats, and using models of 17 and 24 ankyrin repeats. The extension and stiffness of large ankyrin-repeat structures were found to match those predicted by the gating-spring model. Our results suggest that ankyrin repeats of TRPA1 and TRPN1 channels serve as the gating spring for mechanotransduction.     

Large Protein Assemblies Formed by Multivalent Interactions between Cadherin23 and Harmonin Suggest a Stable Anchorage Structure at the Tip Link of Stereocilia

Stereocilia tip links of inner ear hair cells are subjected to constant stretching during hair-bundle deflection, and accordingly are well designed to prevent from being broken by mechanical tensions. The roots of tip links, which couple tip links with the cytoskeleton, supposedly play important roles in withstanding large forces under stimulated conditions. The upper root of the tip link is mainly formed by the cytoplasmic tail of cadherin23 and its actin-anchoring protein harmonin. However, the detailed organization mode of the two proteins that gives rise to a strong upper root remains unclear. Here we show that the exon68-encoded peptide of cadherin23 can either interact with the N-terminal domain (NTD) of harmonin or form a homodimer. We demonstrate that the three harmonin binding sites of cadherin23, namely the NTD-binding motif, the exon68 peptide, and the C-terminal PDZ binding motif, do not synergize with each other in binding to harmonin, instead they facilitate formation of polymeric cadherin23/harmonin complexes. The exon68 peptide can promote the cadherin23/harmonin polymer formation via either binding to harmonin NTD or self-dimerization. We propose that the polymeric cadherin23/harmonin complex formed beneath the upper tip link membranes may serve as part of the stable rootlet structure for anchoring the tip links of stereocilia.     

Rhythmic sensitization of nematocyst discharge in response to vibrational stimuli

Sea anemones capture prey by discharging nematocysts and other cnidae. Discharge of microbasic p-mastigophore (mpm) nematocysts is regulated in part by hair bundle mechanoreceptors on tentacles arising from multicellular complexes consisting of supporting cells and a sensory neuron. Anemone hair bundles detect movements of prey and then sensitize cnidocytes (cnida-containing cells) to discharge mpm nematocysts in response to contact between the prey and tentacle. Data from a simple bioassay based on counting nematocysts discharged into test probes, indicate that approximately twice as many nematocysts discharge into test probes touched to tentacles after sensitization than before sensitization. We here report that sub-second bursts of vibrational stimuli at key frequencies (51, 55, 65, or 74 Hz; Watson GM, Mire P, Hudson RR. 1998. J Exp Zool 281:582-593) sensitize discharge for at least 90 sec. Very few complete cycles of vibration are sufficient to sensitize discharge. However, as the number of cycles of vibration is increased, discharge is sensitized in rhythmic patterns. Computer analysis of the data by fast Fourier transforms indicates discharge to vibrations at 65 Hz is sensitized every 6.75 cycles. At 51 Hz discharge is sensitized every 2.00 cycles. At 74 Hz, discharge is sensitized in a polyrhythm occurring every 4.26, 3.76, 2.46, and 2. 10 cycles, respectively. At 55 Hz, discharge is sensitized in a polyrhythm occurring every 6.09, 3.20, 2.91, and 2.0 cycles, respectively. Apparently, cells in the neuronal pathway interconnecting anemone hair bundles with cnidocytes count cycles of vibration and then sensitize discharge or not according to the tally. J. Exp. Zool. 286:262-269, 2000.     

Wnt signaling mediates reorientation of outer hair cell stereociliary bundles in the mammalian cochlea

In the mammalian cochlea, stereociliary bundles located on mechanosensory hair cells within the sensory epithelium are unidirectionally oriented. Development of this planar polarity is necessary for normal hearing as stereociliary bundles are only sensitive to vibrations in a single plane; however, the mechanisms governing their orientation are unknown. We report that Wnt signaling regulates the development of unidirectional stereociliary bundle orientation. In vitro application of Wnt7a protein or inhibitors of Wnt signaling, secreted Frizzled-related protein 1 or Wnt inhibitory factor 1, disrupts bundle orientation. Moreover, Wnt7a is expressed in a pattern consistent with a role in the polarization of the developing stereociliary bundles. We propose that Wnt signaling across the region of developing outer hair cells gives rise to planar polarity in the mammalian cochlea.     

Usher syndrome type I and the differentiation of inner ear sensory cells' hair bundles

Defects in myosin VIIa, the PDZ-domain-containing protein harmonin, cadherin 23, protocadherin 15, and the putative scaffolding protein sans, underlie five genetic forms of Usher syndrome type I (USH1), the most frequent cause of hereditary deafness-blindness in humans. Mice mutants defective for any of these proteins have a severe hearing impairment and display similar inner ear phenotypes characterized by the abnormal spreading of the sensory cells' stereocilia. These are highly specialized mechanoreceptive organelles derived from microvilli, that normally form a well-structured hair bundle at the apex of inner ear sensory cells. All the USH1 proteins, except sans, have been detected in the growing stereocilia. Moreover, biochemical studies have started to unravel the multiple direct molecular interactions between USH1 proteins. In particular, harmonin can bind to the other four USH1 proteins and to F-actin. Finally, cell biology studies have provided the first insights into the functions of these proteins, and revealed that cadherin 23, and probably protocadherin 15 also, are associated with transient lateral links that interconnect growing stereocilia. These connectors play a critical role in the differentiating hair bundle.     

Sound-induced motions of individual cochlear hair bundles

We present motions of individual freestanding hair bundles in an isolated cochlea in response to tonal sound stimulation. Motions were measured from images taken by strobing a light source at the tone frequency. The tips and bases of hair bundles moved a comparable amount, but with a phase difference that increased by 180 degrees with frequency, indicating that distributed fluid properties drove hair bundle motion. Hair bundle rotation increased with frequency to a constant value, and underwent >90 degrees of phase change. The frequency at which the phase of rotation relative to deflection of the bundle base was 60 degrees was comparable to the expected best frequency of each hair cell, and varied inversely with the square of bundle height. The sharpness of tuning of individual hair bundles was comparable to that of hair cell receptor potentials at high sound levels. These results indicate that frequency selectivity at high sound levels in this cochlea is purely mechanical, determined by the interaction of hair bundles with the surrounding fluid. The sharper tuning of receptor potentials at lower sound levels is consistent with the presence of a negative damping, but not a negative stiffness, as an active amplifier in hair bundles.     

PIST regulates the intracellular trafficking and plasma membrane expression of Cadherin 23

Background  

The atypical cadherin protein cadherin 23 (CDH23) is crucial for proper function of retinal photoreceptors and inner ear hair cells. As we obtain more and more information about the specific roles of cadherin 23 in photoreceptors and hair cells, the regulatory mechanisms responsible for the transport of this protein to the plasma membrane are largely unknown.     

High-purity isolation of bullfrog hair bundles and subcellular and topological localization of constituent proteins.

The small number of hair cells in auditory and vestibular organs severely impedes the biochemical characterization of the proteins involved in mechano-electrical transduction. By developing an efficient and clean "twist-off" method of hair bundle isolation, and by devising a sensitive, nonradioactive method to detect minute quantities of protein, we have partially overcome this limitation and have extensively classified the proteins of the bundles. To isolate hair bundles, we glue the saccular macula of the bullfrog to a glass coverslip, expose the tissue to a molten agarose solution, and allow the agarose to solidify to a firm gel. By rotating the gel disk with respect to the fixed macula, we isolate the hair bundles by shearing them at their mechanically weak bases. The plasma membranes of at least 80% of the stereocilia reseal. To visualize the proteins of the hair bundle, we covalently label them with biotin, separate them by SDS-PAGE, and transfer them to a charged nylon membrane. We can detect less than 500 fg of protein by probing the membrane with streptavidin-alkaline phosphatase and detecting the chemiluminescent product from the hydrolysis of the substrate 3-(4-methoxyspiro-(1,2-dioxetane-3,2'-tricyclo-[3.3.1. 1(3.7)]decan)-4-yl) phenyl phosphate (AMPPD). These techniques reveal a distinct constellation of proteins in and associated with hair bundles. Several proteins, such as calmodulin, calbindin, actin, tubulin, and fimbrin, have previously been described. A second class of proteins in the preparation appears to be derived from extracellular sources. Finally, several heretofore undescribed bundle proteins are identified and characterized by their membrane topology, subcellular localization, and glycosidase and protease sensitivities.     

The role of plant villin in the organization of the actin cytoskeleton, cytoplasmic streaming and the architecture of the transvacuolar strand in root hair cells of Hydrocharis

In many types of plant cell, bundles of actin filaments (AFs) are generally involved in cytoplasmic streaming and the organization of transvacuolar strands. Actin cross-linking proteins are believed to arrange AFs into the bundles. In root hair cells of Hydrocharis dubia (Blume) Baker, a 135-kDa polypeptide cross-reacted with an antiserum against a 135-kDa actin-bundling protein (135-ABP), a villin homologue, isolated from lily pollen tubes. Immunofluorescence microscopy revealed that the 135-kDa polypeptide co-localized with AF bundles in the transvacuolar strand and in the sub-cortical region of the cells. Microinjection of antiserum against 135-ABP into living root hair cells induced the disappearance of the transvacuolar strand. Concomitantly, thick AF bundles in the transvacuolar strand dispersed into thin bundles. In the root hair cells, AFs showed uniform polarity in the bundles, which is consistent with the in-vitro activity of 135-ABP. These results suggest that villin is a factor responsible for bundling AFs in root hair cells as well as in pollen tubes, and that it plays a key role in determining the direction of cytoplasmic streaming in these cells. Received: 16 September 1999 / Accepted: 3 December 1999