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1.  Harmonin-b, an actin-binding scaffold protein, is involved in the adaptation of mechanoelectrical transduction by sensory hair cells 
Pflugers Archiv   2009;459(1):115-130.
We assessed the involvement of harmonin-b, a submembranous protein containing PDZ domains, in the mechanoelectrical transduction machinery of inner ear hair cells. Harmonin-b is located in the region of the upper insertion point of the tip link that joins adjacent stereocilia from different rows and that is believed to gate transducer channel(s) located in the region of the tip link's lower insertion point. In Ush1cdfcr-2J/dfcr-2J mutant mice defective for harmonin-b, step deflections of the hair bundle evoked transduction currents with altered speed and extent of adaptation. In utricular hair cells, hair bundle morphology and maximal transduction currents were similar to those observed in wild-type mice, but adaptation was faster and more complete. Cochlear outer hair cells displayed reduced maximal transduction currents, which may be the consequence of moderate structural anomalies of their hair bundles. Their adaptation was slower and displayed a variable extent. The latter was positively correlated with the magnitude of the maximal transduction current, but the cells that showed the largest currents could be either hyperadaptive or hypoadaptive. To interpret our observations, we used a theoretical description of mechanoelectrical transduction based on the gating spring theory and a motor model of adaptation. Simulations could account for the characteristics of transduction currents in wild-type and mutant hair cells, both vestibular and cochlear. They led us to conclude that harmonin-b operates as an intracellular link that limits adaptation and engages adaptation motors, a dual role consistent with the scaffolding property of the protein and its binding to both actin filaments and the tip link component cadherin-23.
Electronic supplementary material
The online version of this article (doi:10.1007/s00424-009-0711-x) contains supplementary material, which is available to authorized users.
doi:10.1007/s00424-009-0711-x
PMCID: PMC2767239  PMID: 19756723
Cochlea; Hair bundle; Vestibule; Harmonin; Mechanoelectrical transduction; Adaptation; Hair cell; Vestibular system
2.  Twinfilin 2 regulates actin filament lengths in cochlear stereocilia 
Inner ear sensory hair cells convert mechanical stimuli into electrical signals. This conversion happens in the exquisitely mechanosensitive hair bundle that protrudes from the cell's apical surface. In mammals, cochlear hair bundles are composed of 50-100 actin-filled stereocilia, which are organized in three rows in a staircase fashion. Stereocilia actin filaments are uniformly oriented with their barbed ends toward stereocilia tips. During development, the actin core of each stereocilium undergoes elongation due to addition of actin monomers to the barbed ends of the filaments. Here we show that in the mouse cochlea the barbed end capping protein twinfilin 2 is present at the tips of middle and short rows of stereocilia from postnatal day (P) 5 onward, which correlates with a time period when these rows stop growing. The tall stereocilia rows, which do not display twinfilin 2 at their tips, continue to elongate between P5 and P15. When we expressed twinfilin 2 in LLC/PK1-CL4 (CL4) cells, we observed a reduction of espin-induced microvilli length, pointing to a potent function of twinfilin 2 in suppressing the elongation of actin filaments. Overexpression of twinfilin 2 in cochlear inner hair cells resulted in a significant reduction of stereocilia length. Our results suggest that twinfilin 2 plays a role in the regulation of stereocilia elongation by restricting excessive elongation of the shorter row stereocilia thereby maintaining the mature staircase architecture of cochlear hair bundles.
doi:10.1523/JNEUROSCI.2782-09.2009
PMCID: PMC2823077  PMID: 19955359
Auditory; Cochlea; Cytoskeleton; Actin; Mechanosensory; Cochlea
3.  Kinocilia mediate mechanosensitivity in developing zebrafish hair cells 
Developmental cell  2012;23(2):329-341.
SUMMARY
Mechanosensitive cilia are vital to signaling and development across many species. In sensory hair cells, sound and movement are transduced by apical hair bundles. Each bundle is comprised of a single primary cilium (kinocilium) flanked by multiple rows of actin-filled projections (stereocilia). Extracellular tip links that interconnect stereocilia are thought to gate mechanosensitive channels. In contrast to stereocilia, kinocilia are not critical for hair-cell mechanotransduction. However, by sequentially imaging the structure of hair bundles and mechanosensitivity of individual lateral-line hair cells in vivo, we uncovered a central role for kinocilia in mechanosensation during development. Our data demonstrate that nascent hair cells require kinocilia and kinocilial links for mechanosensitivity. Although nascent hair bundles have correct planar polarity, the polarity of their responses to mechanical stimuli is initially reversed. Later in development, a switch to correctly polarized mechanosensitivity coincides with the formation of tip links and the onset of tip link-dependent mechanotransduction.
doi:10.1016/j.devcel.2012.05.022
PMCID: PMC3426295  PMID: 22898777
4.  The Actin-Binding Proteins Eps8 and Gelsolin Have Complementary Roles in Regulating the Growth and Stability of Mechanosensory Hair Bundles of Mammalian Cochlear Outer Hair Cells 
PLoS ONE  2014;9(1):e87331.
Sound transduction depends upon mechanosensitive channels localized on the hair-like bundles that project from the apical surface of cochlear hair cells. Hair bundles show a stair-case structure composed of rows of stereocilia, and each stereocilium contains a core of tightly-packed and uniformly-polarized actin filaments. The growth and maintenance of the stereociliary actin core are dynamically regulated. Recently, it was shown that the actin-binding protein gelsolin is expressed in the stereocilia of outer hair cells (OHCs) and in its absence they become long and straggly. Gelsolin is part of a whirlin scaffolding protein complex at the stereocilia tip, which has been shown to interact with other actin regulatory molecules such as Eps8. Here we investigated the physiological effects associated with the absence of gelsolin and its possible overlapping role with Eps8. We found that, in contrast to Eps8, gelsolin does not affect mechanoelectrical transduction during immature stages of development. Moreover, OHCs from gelsolin knockout mice were able to mature into fully functional sensory receptors as judged by the normal resting membrane potential and basolateral membrane currents. Mechanoelectrical transducer current in gelsolin-Eps8 double knockout mice showed a profile similar to that observed in the single mutants for Eps8. We propose that gelsolin has a non-overlapping role with Eps8. While Eps8 is mainly involved in the initial growth of stereocilia in both inner hair cells (IHCs) and OHCs, gelsolin is required for the maintenance of mature hair bundles of low-frequency OHCs after the onset of hearing.
doi:10.1371/journal.pone.0087331
PMCID: PMC3903700  PMID: 24475274
5.  Molecular Remodeling of Tip Links Underlies Mechanosensory Regeneration in Auditory Hair Cells 
PLoS Biology  2013;11(6):e1001583.
Backscatter scanning electron microscopy and conventional whole cell patch-clamp experiments reveal a two-step mechanism for the regeneration of tip links, the crucial element of mechanotransduction machinery in the hair cells of the inner ear.
Sound detection by inner ear hair cells requires tip links that interconnect mechanosensory stereocilia and convey force to yet unidentified transduction channels. Current models postulate a static composition of the tip link, with protocadherin 15 (PCDH15) at the lower and cadherin 23 (CDH23) at the upper end of the link. In terminally differentiated mammalian auditory hair cells, tip links are subjected to sound-induced forces throughout an organism's life. Although hair cells can regenerate disrupted tip links and restore hearing, the molecular details of this process are unknown. We developed a novel implementation of backscatter electron scanning microscopy to visualize simultaneously immuno-gold particles and stereocilia links, both of only a few nanometers in diameter. We show that functional, mechanotransduction-mediating tip links have at least two molecular compositions, containing either PCDH15/CDH23 or PCDH15/PCDH15. During regeneration, shorter tip links containing nearly equal amounts of PCDH15 at both ends appear first. Whole-cell patch-clamp recordings demonstrate that these transient PCDH15/PCDH15 links mediate mechanotransduction currents of normal amplitude but abnormal Ca2+-dependent decay (adaptation). The mature PCDH15/CDH23 tip link composition is re-established later, concomitant with complete recovery of adaptation. Thus, our findings provide a molecular mechanism for regeneration and maintenance of mechanosensory function in postmitotic auditory hair cells and could help identify elusive components of the mechanotransduction machinery.
Author Summary
The inner ear detects sound when stereocilia, the mechanosensory projections on the apical surface of the hair cells, are deflected and tug on tiny extracellular tip links. These links interconnect stereocilia and convey forces to the mechanosensitive transduction channels. Current models postulate a static composition of the tip link with protocadherin 15 (PCDH15) at the link's bottom end and cadherin 23 (CDH23) at the upper end. Tip links are subjected to substantial sound-induced forces. Although hair cells can renew (regenerate) disrupted tip links and restore hearing, the molecular details of this process are unknown. Our study provides mechanistic insight into tip link regeneration. We used backscatter scanning electron microscopy to monitor the distribution of immuno-gold labeled molecular components of the tip links during their re-formation and a conventional whole-cell patch-clamp technique to follow the concomitant recovery of mechano-electrical transduction. According to our data, the mechanotransduction machinery is initially re-established by the formation of functional (mechanotransduction-mediating) links of a previously unknown composition, PCDH15–PCDH15. Transition to the PCDH15–CDH23 composition underlies final maturation of mechanotransduction. This two-step mechanism of tip link regeneration was unexpected. As tip links are continuously stressed by loud sounds and regenerated throughout an organism's life, we provide a plausible molecular mechanism for the life-long maintenance of mechanosensory function in nonregenerating cochlear hair cells.
doi:10.1371/journal.pbio.1001583
PMCID: PMC3679001  PMID: 23776407
6.  Fast adaptation and Ca2+-sensitivity of the mechanotransducer require myosin-XVa in inner but not outer cochlear hair cells 
In inner ear hair cells, activation of mechotransduction channels is followed by extremely rapid deactivation that depends on the influx of Ca2+ through these channels. Although the molecular mechanisms of this “fast” adaptation are largely unknown, the predominant models assume Ca2+ sensitivity as an intrinsic property of yet unidentified mechanotransduction channels. Here we examined mechanotransduction in the hair cells of young postnatal shaker 2 mice (Myo15sh2/sh2). These mice have no functional myosin-XVa, which is critical for normal growth of mechanosensory stereocilia of hair cells. Although stereocilia of both inner and outer hair cells of Myo15sh2/sh2 mice lack myosin-XVa and are abnormally short, these cells have dramatically different hair bundle morphology. Myo15sh2/sh2 outer hair cells retain a “staircase” arrangement of the abnormally short stereocilia and prominent tip links. Myo15sh2/sh2 inner hair cells do not have obliquely oriented tip links and their mechanosensitivity is mediated exclusively by “top-to-top” links between equally short stereocilia. In both inner and outer hair cells of Myo15sh2/sh2 mice, we found mechanotransduction responses with a normal “wild type” amplitude and speed of activation. Surprisingly, only outer hair cells exhibit fast adaptation and sensitivity to extracellular Ca2+. In Myo15sh2/sh2 inner hair cells, fast adaptation is disrupted and the transduction current is insensitive to extracellular Ca2+. We conclude that the Ca2+-sensitivity of the mechanotransduction channels and the fast adaptation require a structural environment that is dependent on myosin-XVa and is disrupted in Myo15sh2/sh2 inner hair cells, but not in Myo15sh2/sh2 outer hair cells.
doi:10.1523/JNEUROSCI.4566-08.2009
PMCID: PMC2702482  PMID: 19339598
hearing; mechano-electrical transduction; cochlea; shaker 2; non-syndromic deafness; DFNB3
7.  Kinematic analysis of shear displacement as a means for operating mechanotransduction channels in the contact region between adjacent stereocilia of mammalian cochlear hair cells. 
In sensory hair cells of the cochlea, deflection of the stereociliary bundle results in direct mechanical gating of mechanoelectrical transduction channels, a function generally attributed to the tip link running between the tips of short stereocilia and the sides of adjacent taller ones. However, immunocytochemical experiments indicate that the channels may not be associated with the tip link but occur just below it in a region of contact between the stereocilia. To determine whether transduction channels in this location could be operated during physiologically appropriate deflections as effectively by shear displacement as if they were associated with the tip link, a two dimensional kinematic analysis of relative motion between stereocilia has been performed assuming contact between stereocilia is maintained during deflection. Bundle geometry and dimensions were determined from transmission electron micrographs of hair cells from several frequency locations between 0.27 and 13.00 kHz in the guinea-pig cochlea. The analysis indicates that for a 10 nm deflection of the tallest stereocilia of both inner and outer hair cells, i.e. within the range of the maximum sensitivity of mammalian hair bundles, the average shear displacement in the contact region would be 1.6 nm, but that it increases systematically towards higher frequency regions for outer hair cells. This displacement is comparable in magnitude to tip-link elongation for individual stereociliary pairs.
PMCID: PMC1688222  PMID: 9061959
8.  Forces between clustered stereocilia minimize friction in the ear on a subnanometre scale 
Nature  2011;474(7351):376-379.
The detection of sound begins when energy derived from an acoustic stimulus deflects the hair bundles atop hair cells1. As hair bundles move, the viscous friction between stereocilia and the surrounding liquid poses a fundamental physical challenge to the ear’fs high sensitivity and sharp frequency selectivity. Part of the solution to this problem lies in the active process that uses energy for frequency-selective sound amplification2,3. Here we demonstrate that a complementary part of the solution involves the fluid-structure interaction between stereocilia and the liquid within the hair bundle. Using force measurement on a dynamically scaled model, finite-element analysis, analytical estimation of hydrodynamic forces, stochastic simulation, and high-resolution interferometric measurement of hair bundles, we characterize the origin and magnitude of the forces between individual stereocilia during small hair-bundle deflections. We find that the close apposition of stereocilia effectively immobilizes the liquid between them, which reduces the drag and suppresses the relative squeezing but not the sliding mode of stereociliary motion. The obliquely oriented tip links couple the mechanotransduction channels to this least dissipative coherent mode, whereas the elastic horizontal top connectors that stabilize the structure further reduce the drag. As measured from the distortion products associated with channel gating at physiological stimulation amplitudes of tens of nanometres, the balance of viscous and elastic forces in a hair bundle permits a relative mode of motion between adjacent stereocilia that encompasses only a fraction of a nanometre. A combination of high-resolution experiments and detailed numerical modelling of fluid-structure interactions reveals the physical principles behind the basic structural features of hair bundles and shows quantitatively how these organelles are adapted to the needs of sensitive mechanotransduction.
doi:10.1038/nature10073
PMCID: PMC3150833  PMID: 21602823
9.  Mutations of the Mouse ELMO Domain Containing 1 Gene (Elmod1) Link Small GTPase Signaling to Actin Cytoskeleton Dynamics in Hair Cell Stereocilia 
PLoS ONE  2012;7(4):e36074.
Stereocilia, the modified microvilli projecting from the apical surfaces of the sensory hair cells of the inner ear, are essential to the mechanoelectrical transduction process underlying hearing and balance. The actin-filled stereocilia on each hair cell are tethered together by fibrous links to form a highly patterned hair bundle. Although many structural components of hair bundles have been identified, little is known about the signaling mechanisms that regulate their development, morphology, and maintenance. Here, we describe two naturally occurring, allelic mutations that result in hearing and balance deficits in mice, named roundabout (rda) and roundabout-2J (rda2J). Positional cloning identified both as mutations of the mouse ELMO domain containing 1 gene (Elmod1), a poorly characterized gene with no previously reported mutant phenotypes. The rda mutation is a 138 kb deletion that includes exons 1–5 of Elmod1, and rda2J is an intragenic duplication of exons 3–8 of Elmod1. The deafness associated with these mutations is caused by cochlear hair cell dysfunction, as indicated by conspicuous elongations and fusions of inner hair cell stereocilia and progressive degeneration of outer hair cell stereocilia. Mammalian ELMO-family proteins are known to be involved in complexes that activate small GTPases to regulate the actin cytoskeleton during phagocytosis and cell migration. ELMOD1 and ELMOD2 recently were shown to function as GTPase-activating proteins (GAPs) for the Arf family of small G proteins. Our finding connecting ELMOD1 deficiencies with stereocilia dysmorphologies thus establishes a link between the Ras superfamily of small regulatory GTPases and the actin cytoskeleton dynamics of hair cell stereocilia.
doi:10.1371/journal.pone.0036074
PMCID: PMC3338648  PMID: 22558334
10.  The deaf mouse mutant whirler suggests a role for whirlin in actin filament dynamics and stereocilia development 
Stereocilia, finger-like projections forming the hair bundle on the apical surface of sensory hair cells in the cochlea, are responsible for mechanosensation and ultimately the perception of sound. The actin cytoskeleton of the stereocilia contains hundreds of tightly cross-linked parallel actin filaments in a paracrystalline array and it is vital for their function. Although several genes have been identified and associated with stereocilia development, the molecular mechanisms responsible for stereocilia growth, maintenance and organisation of the hair bundle have not been fully resolved. Here we provide further characterisation of the stereocilia of the whirler mouse mutant. We found that a lack of whirlin protein in whirler mutants results in short stereocilia with larger diameters without a corresponding increase in the number of actin filaments in inner hair cells. However, a decrease in the actin filament packing density was evident in the whirler mutant. The electron-density at the tip of each stereocilium was markedly patchy and irregular in the whirler mutants compared with a uniform band in controls. The outer hair cell stereocilia of the whirler homozygote also showed an increase in diameter and variable heights within bundles. The number of outer hair cell stereocilia was significantly reduced and the centre-to-centre spacing between the stereocilia was greater than in the wildtype. Our findings suggest that whirlin plays an important role in actin filament packing and dynamics during postnatal stereocilium elongation.
doi:10.1002/cm.20199
PMCID: PMC2682331  PMID: 17326148
stereocilia; actin filaments; deafness; whirlin; hair cells; inner ear; organ of Corti; cochlea
11.  Roles of the Espin Actin-Bundling Proteins in the Morphogenesis and Stabilization of Hair Cell Stereocilia Revealed in CBA/CaJ Congenic Jerker Mice 
PLoS Genetics  2011;7(3):e1002032.
Hearing and vestibular function depend on mechanosensory staircase collections of hair cell stereocilia, which are produced from microvillus-like precursors as their parallel actin bundle scaffolds increase in diameter and elongate or shorten. Hair cell stereocilia contain multiple classes of actin-bundling protein, but little is known about what each class contributes. To investigate the roles of the espin class of actin-bundling protein, we used a genetic approach that benefited from a judicious selection of mouse background strain and an examination of the effects of heterozygosity. A congenic jerker mouse line was prepared by repeated backcrossing into the inbred CBA/CaJ strain, which is known for excellent hearing and minimal age-related hearing loss. We compared stereocilia in wild-type CBA/CaJ mice, jerker homozygotes that lack espin proteins owing to a frameshift mutation in the espin gene, and jerker heterozygotes that contain reduced espin levels. The lack of espins radically impaired stereociliary morphogenesis, resulting in stereocilia that were abnormally thin and short, with reduced differential elongation to form a staircase. Mean stereociliary diameter did not increase beyond ∼0.10–0.14 µm, making stereocilia ∼30%–60% thinner than wild type and suggesting that they contained ∼50%–85% fewer actin filaments. These characteristics indicate a requirement for espins in the appositional growth and differential elongation of the stereociliary parallel actin bundle and fit the known biological activities of espins in vitro and in transfected cells. The stereocilia of jerker heterozygotes showed a transient proximal-distal tapering suggestive of haploinsufficiency and a slowing of morphogenesis that revealed previously unrecognized assembly steps and intermediates. The lack of espins also led to a region-dependent degeneration of stereocilia involving shortening and collapse. We conclude that the espin actin-bundling proteins are required for the assembly and stabilization of the stereociliary parallel actin bundle.
Author Summary
Stereocilia are the fingerlike projections of inner ear hair cells that detect sound and motion. Stereocilia grow to specific lengths and diameters and form staircase-like arrays. The changes in size appear to be driven by matching alterations in the dimensions of an underlying molecular scaffold consisting of a bundle of actin filaments cross-linked by actin-bundling proteins. To elucidate the roles of the espin actin-bundling proteins in hair cell stereocilia, we carry out an in-depth accounting of stereociliary size and shape in the jerker mutant mouse, which lacks the espin proteins because of a mutation in the espin gene. We examine a new and improved jerker mouse with a genetic background known for high-quality lifelong hearing. We find that, in the absence of espins, stereocilia do not increase in diameter or complete their elongation, but instead bend, shorten, and disappear. Although the specifics vary according to inner ear region, the stereociliary defects are profound and can readily account for the deafness and balance problems of jerker mice and humans with certain espin gene mutations. Even reducing espin levels by one-half leads to temporary defects in stereociliary diameter. Thus, espins play crucial roles in the formation and maintenance of hair cell stereocilia.
doi:10.1371/journal.pgen.1002032
PMCID: PMC3063760  PMID: 21455486
12.  Simulation of the Response of the Inner Hair Cell Stereocilia Bundle to an Acoustical Stimulus 
PLoS ONE  2011;6(3):e18161.
Mammalian hearing relies on a cochlear hydrodynamic sensor embodied in the inner hair cell stereocilia bundle. It is presumed that acoustical stimuli induce a fluid shear-driven motion between the tectorial membrane and the reticular lamina to deflect the bundle. It is hypothesized that ion channels are opened by molecular gates that sense tension in tip-links, which connect adjacent stepped rows of stereocilia. Yet almost nothing is known about how the fluid and bundle interact. Here we show using our microfluidics model how each row of stereocilia and their associated tip links and gates move in response to an acoustical input that induces an orbital motion of the reticular lamina. The model confirms the crucial role of the positioning of the tectorial membrane in hearing, and explains how this membrane amplifies and synchronizes the timing of peak tension in the tip links. Both stereocilia rotation and length change are needed for synchronization of peak tip link tension. Stereocilia length change occurs in response to accelerations perpendicular to the oscillatory fluid shear flow. Simulations indicate that nanovortices form between rows to facilitate diffusion of ions into channels, showing how nature has devised a way to solve the diffusive mixing problem that persists in engineered microfluidic devices.
doi:10.1371/journal.pone.0018161
PMCID: PMC3069064  PMID: 21483823
13.  Gelsolin Plays a Role in the Actin Polymerization Complex of Hair Cell Stereocilia 
PLoS ONE  2010;5(7):e11627.
A complex of proteins scaffolded by the PDZ protein, whirlin, reside at the stereocilia tip and are critical for stereocilia development and elongation. We have shown that in outer hair cells (OHCs) whirlin is part of a larger complex involving the MAGUK protein, p55, and protein 4.1R. Whirlin interacts with p55 which is expressed exclusively in outer hair cells (OHC) in both the long stereocilia that make up the stereocilia bundle proper as well as surrounding shorter microvilli that will eventually regress. In erythrocytes, p55 forms a tripartite complex with protein 4.1R and glycophorin C promoting the assembly of actin filaments and the interaction of whirlin with p55 indicates that it plays a similar role in OHC stereocilia. However, the components directly involved in actin filament regulation in stereocilia are unknown. We have investigated additional components of the whirlin interactome by identifying interacting partners to p55. We show that the actin capping and severing protein, gelsolin, is a part of the whirlin complex. Gelsolin is detected in OHC where it localizes to the tips of the shorter rows but not to the longest row of stereocilia and the pattern of localisation at the apical hair cell surface is strikingly similar to p55. Like p55, gelsolin is ablated in the whirler and shaker2 mutants. Moreover, in a gelsolin mutant, stereocilia in the apex of the cochlea become long and straggly indicating defects in the regulation of stereocilia elongation. The identification of gelsolin provides for the first time a link between the whirlin scaffolding protein complex involved in stereocilia elongation and a known actin regulatory molecule.
doi:10.1371/journal.pone.0011627
PMCID: PMC2905391  PMID: 20661277
14.  Harmonin mutations cause mechanotransduction defects in cochlear hair cells 
Neuron  2009;62(3):375-387.
In hair cells, mechanotransduction channels are gated by tip links, the extracellular filaments that consist of cadherin 23 (CDH23) and protocadherin 15 (PCDH15) and connect the stereocilia of each hair cell. However, which molecules mediate cadherin function at tip links is not known. Here we show that the PDZ-domain protein harmonin is a component of the upper tip-link density (UTLD), where CDH23 inserts into the stereociliary membrane. Harmonin domains that mediate interactions with CDH23 and F-actin control harmonin localization in stereocilia and are necessary for normal hearing. In mice expressing a mutant harmonin protein that prevents UTLD formation, the sensitivity of hair bundles to mechanical stimulation is reduced. We conclude that harmonin is a UTLD component and contributes to establishing the sensitivity of mechanotransduction channels to displacement.
doi:10.1016/j.neuron.2009.04.006
PMCID: PMC2691393  PMID: 19447093
harmonin; Usher Syndrome; mechanotransduction; stereocilia; hair cells
15.  β-actin and fascin-2 cooperate to maintain stereocilia length 
Stereocilia are actin-based protrusions on auditory sensory hair cells that are deflected by sound waves to initiate the conversion of mechanical energy to neuronal signals. Stereocilia maintenance is essential because auditory hair cells are not renewed in mammals. This process requires both β-actin and γ-actin as knockout mice lacking either isoform develop distinct stereocilia pathology during aging. In addition, stereocilia integrity may hinge on immobilizing actin, which outside of a small region at stereocilia tips turns over with a very slow, months-long half-life. Here, we establish that β-actin and the actin crosslinking protein fascin-2 cooperate to maintain stereocilia length and auditory function. We observed that mice expressing mutant fascin-2 (p.R109H) or mice lacking β-actin share a common phenotype including progressive, high-frequency hearing loss together with shortening of a defined subset of stereocilia in the hair cell bundle. Fascin-2 binds β-actin and γ-actin filaments with similar affinity in vitro and fascin-2 does not depend on β-actin for localization in vivo. Nevertheless, double mutant mice lacking β-actin and expressing fascin-2 p.R109H have a more severe phenotype suggesting that each protein has a different function in a common stereocilia maintenance pathway. Since the fascin-2 p.R109H mutant binds but fails to efficiently crosslink actin filaments, we propose that fascin-2 crosslinks function to slow actin depolymerization at stereocilia tips to maintain stereocilia length.
doi:10.1523/JNEUROSCI.0238-13.2013
PMCID: PMC3718021  PMID: 23658152
16.  The R109H variant of fascin-2, a developmentally regulated actin crosslinker in hair-cell stereocilia, underlies early-onset hearing loss of DBA/2J mice 
The quantitative trait locus ahl8 is a key contributor to the early-onset, age-related hearing loss of DBA/2J mice. A non-synonymous nucleotide substitution in the mouse fascin-2 gene (Fscn2) is responsible for this phenotype, confirmed by wild-type BAC transgene rescue of hearing loss in DBA/2J mice. In chickens and mice, FSCN2 protein is abundant in hair-cell stereocilia, the actin-rich structures comprising the mechanically sensitive hair bundle, and is concentrated towards stereocilia tips of the bundle's longest stereocilia. FSCN2 expression increases when these stereocilia differentially elongate, suggesting that FSCN2 controls filament growth, stiffens exposed stereocilia, or both. Because ahl8 accelerates hearing loss only in the presence of mutant cadherin 23, a component of hair-cell tip links, mechanotransduction and actin crosslinking must be functionally interrelated.
doi:10.1523/JNEUROSCI.1541-10.2010
PMCID: PMC2922854  PMID: 20660251
Hair cells; stereocilia; actin; actin crosslinkers; deafness; mutant mice
17.  Localization of inner hair cell mechanotransducer channels using high speed calcium imaging 
Nature neuroscience  2009;12(5):553-558.
Hair cells detect vibrations of their stereociliary bundle by activation of mechanically-sensitive transducer (MT) channels. Although evidence suggests the MT channels are near the stereociliary tops and are opened by force imparted by tip links connecting contiguous stereocilia, the exact channel site remains controversial. Fast confocal imaging of fluorescence changes reflecting calcium entry during bundle stimulation was used to localize MT channels. Calcium signals were visible in single stereocilia of rat cochlear hair cells and were up to ten times larger and faster in the second and third stereociliary rows than in the tallest first row. The number of functional stereocilia was proportional to MT current amplitude indicating about two channels/stereocilium. Comparable results were obtained in outer hair cells. The observations, supported by theoretical simulations, suggest there are no functional MT channels in first row stereocilia and imply the channels are present only at the bottom of the tip links.
doi:10.1038/nn.2295
PMCID: PMC2712647  PMID: 19330002
18.  The role of transmembrane channel–like proteins in the operation of hair cell mechanotransducer channels 
The Journal of General Physiology  2013;142(5):493-505.
Sound stimuli elicit movement of the stereocilia that make up the hair bundle of cochlear hair cells, putting tension on the tip links connecting the stereocilia and thereby opening mechanotransducer (MT) channels. Tmc1 and Tmc2, two members of the transmembrane channel–like family, are necessary for mechanotransduction. To assess their precise role, we recorded MT currents elicited by hair bundle deflections in mice with null mutations of Tmc1, Tmc2, or both. During the first postnatal week, we observed a normal MT current in hair cells lacking Tmc1 or Tmc2; however, in the absence of both isoforms, we recorded a large MT current that was phase-shifted 180°, being evoked by displacements of the hair bundle away from its tallest edge rather than toward it as in wild-type hair cells. The anomalous MT current in hair cells lacking Tmc1 and Tmc2 was blocked by FM1-43, dihydrostreptomycin, and extracellular Ca2+ at concentrations similar to those that blocked wild type. MT channels in the double knockouts carried Ca2+ with a lower permeability than wild-type or single mutants. The MT current in double knockouts persisted during exposure to submicromolar Ca2+, even though this treatment destroyed the tip links. We conclude that the Tmc isoforms do not themselves constitute the MT channel but are essential for targeting and interaction with the tip link. Changes in the MT conductance and Ca2+ permeability observed in the absence of Tmc1 mutants may stem from loss of interaction with protein partners in the transduction complex.
doi:10.1085/jgp.201311068
PMCID: PMC3813385  PMID: 24127526
19.  Fate of Mammalian Cochlear Hair Cells and Stereocilia after Loss of the Stereocilia 
Cochlear hair cells transduce mechanical stimuli into electrical activity. The site of hair cell transduction is the hair bundle, an array of stereocilia with different height arranged in a staircase. Tip links connect the apex of each stereocilium to the side of its taller neighbor. The hair bundle and tip links of hair cells are susceptible to acoustic trauma and ototoxic drugs. It has been shown that hair cells in lower vertebrates and in the mammalian vestibular system may survive bundle loss and undergo self-repair of the stereocilia. Our goals were to determine whether cochlear hair cells could survive the trauma and whether the tip link and/or the hair bundle could be regenerated. We simulated the acoustic trauma-induced tip link damage or stereociliary loss by disrupting tip links or ablating the hair bundles in the cultured organ of Corti from neonatal gerbils. Hair-cell fate and stereociliary morphology and function were examined using confocal and scanning electron microscopies and electrophysiology. Most bundleless hair cells survived and developed for about 2 weeks. However, no spontaneous hair-bundle regeneration was observed. When tip links were ruptured, repair of tip links and restoration of mechanotransduction were observed in less than 24 hours. Our study suggests that the dynamic nature of the hair cell's transduction apparatus is retained despite the fact that regeneration of the hair bundle is lost in mammalian cochlear hair cells.
doi:10.1523/JNEUROSCI.3231-09.2009
PMCID: PMC2795320  PMID: 19955380
hair cells; repair; stereocilia; tip links; gerbil; culture
20.  The Very Large G-Protein-Coupled Receptor VLGR1: A Component of the Ankle Link Complex Required for the Normal Development of Auditory Hair Bundles 
Sensory hair bundles in the inner ear are composed of stereocilia that can be interconnected by a variety of different link types, including tip links, horizontal top connectors, shaft connectors, and ankle links. The ankle link antigen is an epitope specifically associated with ankle links and the calycal processes of photoreceptors in chicks. Mass spectrometry and immunoblotting were used to identify this antigen as the avian ortholog of the very large G-protein-coupled receptor VLGR1, the product of the Usher syndrome USH2C (Mass1) locus. Like ankle links, Vlgr1 is expressed transiently around the base of developing hair bundles in mice. Ankle links fail to form in the cochleae of mice carrying a targeted mutation in Vlgr1 (Vlgr1/del7TM), and the bundles become disorganized just after birth. FM1-43 [N-(3-triethylammonium)propyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide] dye loading and whole-cell recordings indicate mechanotransduction is impaired in cochlear, but not vestibular, hair cells of early postnatal Vlgr1/del7TM mutant mice. Auditory brainstem recordings and distortion product measurements indicate that these mice are severely deaf by the third week of life. Hair cells from the basal half of the cochlea are lost in 2-month-old Vlgr1/del7TM mice, and retinal function is mildly abnormal in aged mutants. Our results indicate that Vlgr1 is required for formation of the ankle link complex and the normal development of cochlear hair bundles.
doi:10.1523/JNEUROSCI.0693-06.2006
PMCID: PMC2682555  PMID: 16775142
cochlea; hair cell; Usher syndrome; knock-out mice; GPCR; retina
21.  An actin molecular treadmill and myosins maintain stereocilia functional architecture and self-renewal 
The Journal of Cell Biology  2004;164(6):887-897.
We have previously shown that the seemingly static paracrystalline actin core of hair cell stereocilia undergoes continuous turnover. Here, we used the same approach of transfecting hair cells with actin–green fluorescent protein (GFP) and espin-GFP to characterize the turnover process. Actin and espin are incorporated at the paracrystal tip and flow rearwards at the same rate. The flux rates (∼0.002–0.04 actin subunits s−1) were proportional to the stereocilia length so that the entire staircase stereocilia bundle was turned over synchronously. Cytochalasin D caused stereocilia to shorten at rates matching paracrystal turnover. Myosins VI and VIIa were localized alongside the actin paracrystal, whereas myosin XVa was observed at the tips at levels proportional to stereocilia lengths. Electron microscopy analysis of the abnormally short stereocilia in the shaker 2 mice did not show the characteristic tip density. We argue that actin renewal in the paracrystal follows a treadmill mechanism, which, together with the myosins, dynamically shapes the functional architecture of the stereocilia bundle.
doi:10.1083/jcb.200310055
PMCID: PMC2172292  PMID: 15024034
hair cells; myosin XVa; myosin VIIa; espin; hearing
22.  Sliding adhesion confers coherent motion to hair cell stereocilia and parallel gating to transduction channels 
When the tip of a hair bundle is deflected by a sensory stimulus, the stereocilia pivot as a unit, producing a shearing displacement between adjacent tips. It is not clear how stereocilia can stick together laterally but still shear. We used dissociated hair cells from the bullfrog saccule and high-speed video imaging to characterize this sliding adhesion. Movement of individual stereocilia was proportional to height, indicating that stereocilia pivot at their basal insertion points. All stereocilia moved by approximately the same angular deflection, and the same motion was observed at 1, 20 and 700 Hz stimulus frequency. Motions were consistent with a geometric model that assumes the stiffness of lateral links holding stereocilia together is >1000 times the pivot stiffness of stereocilia and that these links can slide in the plane of the membrane—in essence, that stereocilia shear without separation. The same motion was observed when bundles were moved perpendicular to the tip links, or when tip links, ankle links and shaft connectors were cut, ruling out these links as the basis for sliding adhesion. Stereocilia rootlets are angled towards the center of the bundle, tending to push stereocilia tips together for small deflections. However, stereocilia remained cohesive for deflections of up to ±35°, ruling out rootlet prestressing as the basis for sliding adhesion. These observations suggest that horizontal top connectors mediate a sliding adhesion. They also indicate that all transduction channels of a hair cell are mechanically in parallel, an arrangement that may enhance amplification in the inner ear.
doi:10.1523/JNEUROSCI.4864-09.2010
PMCID: PMC2932470  PMID: 20610739
stereocilia; hair cell; cell adhesion
23.  MyosinVIIa Interacts with Twinfilin-2 at the Tips of Mechanosensory Stereocilia in the Inner Ear 
PLoS ONE  2009;4(9):e7097.
In vertebrates hearing is dependent upon the microvilli-like mechanosensory stereocilia and their length gradation. The staircase-like organization of the stereocilia bundle is dynamically maintained by variable actin turnover rates. Two unconventional myosins were previously implicated in stereocilia length regulation but the mechanisms of their action remain unknown. MyosinXVa is expressed in stereocilia tips at levels proportional to stereocilia length and its absence produces staircase-like bundles of very short stereocilia. MyosinVIIa localizes to the tips of the shorter stereocilia within bundles, and when absent, the stereocilia are abnormally long. We show here that myosinVIIa interacts with twinfilin-2, an actin binding protein, which inhibits actin polymerization at the barbed end of the filament, and that twinfilin localization in stereocilia overlaps with myosinVIIa. Exogenous expression of myosinVIIa in fibroblasts results in a reduced number of filopodia and promotes accumulation of twinfilin-2 at the filopodia tips. We hypothesize that the newly described interaction between myosinVIIa and twinfilin-2 is responsible for the establishment and maintenance of slower rates of actin turnover in shorter stereocilia, and that interplay between complexes of myosinVIIa/twinfilin-2 and myosinXVa/whirlin is responsible for stereocilia length gradation within the bundle staircase.
doi:10.1371/journal.pone.0007097
PMCID: PMC2743196  PMID: 19774077
24.  MAGI-1, a candidate stereociliary scaffolding protein associates with the tip link component Cadherin 23 
Inner ear hair cell mechanoelectrical transduction is mediated by a largely unidentified multi-protein complex associated with the stereociliary tips of hair bundles. One identified component of tip links, which are the extracellular filamentous connectors implicated in gating the mechanoelectrical transduction channels, is the transmembrane protein Cadherin 23 (Cdh23), more specifically, the hair cell-specific Cdh23(+68) splice variant. Using the intracellular domain of Cdh23(+68) as bait, we identified in a cochlear cDNA library MAGI-1, a membrane-associated guanylate kinase (MAGUK) protein. MAGI-1 binds via its PDZ4 domain to a carboxyl-terminal PDZ binding site on Cdh23. MAGI-1 immunoreactivity was detectable throughout neonatal stereocilia in a similar distribution as Cdh23, and as development proceeded, MAGI-1 occurred in a punctate staining pattern on stereocilia, which was maintained into adulthood. Previous reports suggest that Cdh23 interacts via an internal PDZ binding site with the PDZ1 domain of the stereociliary protein harmonin, and potentially via a weaker binding of its carboxyl-terminus with harmonin’s PDZ2 domain. We propose that MAGI-1 has the ability to replace harmonin’s PDZ2 binding at Cdh23′s carboxyl-terminus. Moreover, the strong interaction between PDZ1 of harmonin and Cdh23 is interrupted by a 35-amino acid insertion in the hair cell-specific Cdh23(+68) splice variant, which puts forward MAGI-1 as an attractive candidate for an intracellular scaffolding partner of this tip link protein. Our results consequently support a role of MAGI-1 in the tip link complex where it could provide a sturdy connection with the cytoskeleton and with other components of the mechanoelectrical transduction complex.
doi:10.1523/JNEUROSCI.3833-08.2008
PMCID: PMC2596868  PMID: 18971469
Cochlea; Hair cell; Inner ear; Mechanotransduction; Protocadherin 15; Usher syndrome
25.  TMHS is an Integral Component of the Mechanotransduction Machinery of Cochlear Hair Cells 
Cell  2012;151(6):1283-1295.
SUMMARY
Hair cells are mechanosensors for the perception of sound, acceleration and fluid motion. Mechanotransduction channels in hair cells are gated by tip links, which connect the stereocilia of a hair cell in the direction of their mechanical sensitivity. The molecular constituents of the mechanotransduction channels of hair cells are not known. Here we show that mechanotransduction is impaired in mice lacking the tetraspan TMHS. TMHS binds to the tip-link component PCDH15 and regulates tip-link assembly, a process that is disrupted by deafness-causing Tmhs mutations. TMHS also regulates transducer channel conductance and is required for fast channel adaptation. TMHS therefore resembles other ion channel regulatory subunits such as the TARPs of AMPA receptors that facilitate channel transport and regulate the properties of pore-forming channel subunits. We conclude that TMHS is an integral component of the hair cells mechanotransduction machinery that functionally couples PCDH15 to the transduction channel.
doi:10.1016/j.cell.2012.10.041
PMCID: PMC3522178  PMID: 23217710

Results 1-25 (586364)