Tectorins crosslink type II collagen fibrils and connect the tectorial membrane to the spiral limbus.

All inner ear organs possess extracellular matrix appendices over the sensory epithelia that are crucial for their proper function. The tectorial membrane (TM) is a gelatinous acellular membrane located above the hearing sensory epithelium and is composed mostly of type II collagen, and α and ß tectorins. TM molecules self-assemble in the endolymph fluid environment, interacting medially with the spiral limbus and distally with the outer hair cell stereocilia. Here, we used immunogold labeling in freeze-substituted mouse cochleae to assess the fine localization of both tectorins in distinct TM regions. We observed that the TM adheres to the spiral limbus through a dense thin matrix enriched in α- and ß-tectorin, both likely bound to the membranes of interdental cells. Freeze-etching images revealed that type II collagen fibrils were crosslinked by short thin filaments (4±1.5nm, width), resembling another collagen type protein, or chains of globular elements (15±3.2nm, diameter). Gold-particles for both tectorins also localized adjacent to the type II collagen fibrils, suggesting that these globules might be composed essentially of α- and ß-tectorins. Finally, the presence of gold-particles at the TM lower side suggests that the outer hair cell stereocilia membrane has a molecular partner to tectorins, probably stereocilin, allowing the physical connection between the TM and the organ of Corti.

Thyroid hormone is required for pruning, functioning and long-term maintenance of afferent inner hair cell synapses.

Functional maturation of afferent synaptic connections to inner hair cells (IHCs) involves pruning of excess synapses formed during development, as well as the strengthening and survival of the retained synapses. These events take place during the thyroid hormone (TH)-critical period of cochlear development, which is in the perinatal period for mice and in the third trimester for humans. Here, we used the hypothyroid Snell dwarf mouse (Pit1(dw)) as a model to study the role of TH in afferent type I synaptic refinement and functional maturation. We observed defects in afferent synaptic pruning and delays in calcium channel clustering in the IHCs of Pit1(dw) mice. Nevertheless, calcium currents and capacitance reached near normal levels in Pit1(dw) IHCs by the age of onset of hearing, despite the excess number of retained synapses. We restored normal synaptic pruning in Pit1(dw) IHCs by supplementing with TH from postnatal day (P)3 to P8, establishing this window as being critical for TH action on this process. Afferent terminals of older Pit1(dw) IHCs showed evidence of excitotoxic damage accompanied by a concomitant reduction in the levels of the glial glutamate transporter, GLAST. Our results indicate that a lack of TH during a critical period of inner ear development causes defects in pruning and long-term homeostatic maintenance of afferent synapses.

Missense mutations in Otopetrin 1 affect subcellular localization and inhibition of purinergic signaling in vestibular supporting cells.

Otopetrin 1 (Otop1) encodes a protein that is essential for the development of otoconia. Otoconia are the extracellular calcium carbonate containing crystals that are important for vestibular mechanosensory transduction of linear motion and gravity. There are two mutant alleles of Otop1 in mice, titled (tlt) and mergulhador (mlh), which result in non-syndromic otoconia agenesis and a consequent balance defect. Biochemically, Otop1 has been shown to modulate purinergic control of intracellular calcium in vestibular supporting cells, which could be one of the mechanisms by which Otop1 participates in the mineralization of otoconia. To understand how tlt and mlh mutations affect the biochemical function of Otop1, we examined the purinergic response of COS7 cells expressing mutant Otop1 proteins, and dissociated sensory epithelial cells from tlt and mlh mice. We also examined the subcellular localization of Otop1 in whole sensory epithelia from tlt and mlh mice. Here we show that tlt and mlh mutations uncouple Otop1 from inhibition of P2Y receptor function. Although the in vitro biochemical function of the Otop1 mutant proteins is normal, in vivo they behave as null alleles. We show that in supporting cells the apical membrane localization of the mutant Otop1 proteins is lost. These data suggest that the tlt and mlh mutations primarily affect the localization of Otop1, which interferes with its ability to interact with other proteins that are important for its cellular and biochemical function.

Swept field laser confocal microscopy for enhanced spatial and temporal resolution in live-cell imaging.

Confocal fluorescence microscopy is a broadly used imaging technique that enhances the signal-to-noise ratio by removing out of focal plane fluorescence. Confocal microscopes come with a variety of modifications depending on the particular experimental goals. Microscopes, illumination pathways, and light collection were originally focused upon obtaining the highest resolution image possible, typically on fixed tissue. More recently, live-cell confocal imaging has gained importance. Since measured signals are often rapid or transient, thus requiring higher sampling rates, specializations are included to enhance spatial and temporal resolution while maintaining tissue viability. Thus, a balance between image quality, temporal resolution, and tissue viability is needed. A subtype of confocal imaging, termed swept field confocal (SFC) microscopy, can image live cells at high rates while maintaining confocality. SFC systems can use a pinhole array to obtain high spatial resolution, similar to spinning disc systems. In addition, SFC imaging can achieve faster rates by using a slit to sweep the light across the entire image plane, thus requiring a single scan to generate an image. Coupled to a high-speed charge-coupled device camera and a laser illumination source, images can be obtained at greater than 1,000 frames per second while maintaining confocality.

Intermolecular autophosphorylation regulates myosin IIIa activity and localization in parallel actin bundles.

Myosin IIIa (Myo3A) transports cargo to the distal end of actin protrusions and contains a kinase domain that is thought to autoregulate its activity. Because Myo3A tends to cluster at the tips of actin protrusions, we investigated whether intermolecular phosphorylation could regulate Myo3A biochemical activity, cellular localization, and cellular function. Inactivation of Myo3A 2IQ kinase domain with the point mutation K50R did not alter maximal ATPase activity, whereas phosphorylation of Myo3A 2IQ resulted in reduced maximal ATPase activity and actin affinity. The rate and degree of Myo3A 2IQ autophosphorylation was unchanged by the presence of actin but was found to be dependent upon Myo3A 2IQ concentration within the range of 0.1 to 1.2 µm, indicating intermolecular autophosphorylation. In cultured cells, we observed that the filopodial tip localization of Myo3A lacking the kinase domain decreased when co-expressed with kinase-active, full-length Myo3A. The cellular consequence of reduced Myo3A tip localization was decreased filopodial density along the cell periphery, identifying a novel cellular function for Myo3A in mediating the formation and stability of actin-based protrusions. Our results suggest that Myo3A motor activity is regulated through a mechanism involving concentration-dependent autophosphorylation. We suggest that this regulatory mechanism plays an essential role in mediating the transport and actin bundle formation/stability functions of Myo3A.

The septate junction protein caspr is required for structural support and retention of KCNQ4 at calyceal synapses of vestibular hair cells.

The afferent innervation contacting the type I hair cells of the vestibular sensory epithelia form distinct calyceal synapses. The apposed presynaptic and postsynaptic membranes at this large area of synaptic contact are kept at a remarkably regular distance. Here, we show by freeze-fracture electron microscopy that a patterned alignment of proteins at the calyceal membrane resembles a type of intercellular junction that is rare in vertebrates, the septate junction (SJ). We found that a core molecular component of SJs, Caspr, colocalizes with the K(+) channel KCNQ4 at the postsynaptic membranes of these calyceal synapses. Immunolabeling and ultrastructural analyses of Caspr knock-out mice reveal that, in the absence of Caspr, the separation between the membranes of the hair cells and the afferent neurons is conspicuously irregular and often increased by an order of magnitude. In these mutants, KCNQ4 fails to cluster at the postsynaptic membrane and appears diffused along the entire calyceal membrane. Our results indicate that a septate-like junction provides structural support to calyceal synaptic contact with the vestibular hair cell and that Caspr is required for the recruitment or retention of KCNQ4 at these synapses.

Regulation of cellular calcium in vestibular supporting cells by otopetrin 1.

Otopetrin 1 (OTOP1) is a multitransmembrane domain protein, which is essential for mineralization of otoconia, the calcium carbonate biominerals required for vestibular function, and the normal sensation of gravity. The mechanism driving mineralization of otoconia is poorly understood, but it has been proposed that supporting cells and a mechanism to maintain high concentrations of calcium are critical. Using Otop1 knockout mice and a utricular epithelial organ culture system, we show that OTOP1 is expressed at the apex of supporting cells and functions to increase cytosolic calcium in response to purinergic agonists, such as adenosine 5'-triphosphate (ATP). This is achieved by blocking mobilization of calcium from intracellular stores in an extracellular calcium-dependent manner and by mediating influx of extracellular calcium. These data support a model in which OTOP1 acts as a sensor of the extracellular calcium concentration near supporting cells and responds to ATP in the endolymph to increase intracellular calcium levels during otoconia mineralization.

Stepwise morphological and functional maturation of mechanotransduction in rat outer hair cells.

Inner ear mechanosensory hair cells convert mechanical vibrations into electrical signals via the coordinated interaction of multiple proteins precisely positioned within the sensory hair bundle. Present work identifies the time course for the acquisition and maturation of mechanoelectric transduction (MET) in rat cochlea outer hair cells maintained in organotypic cultures. A spatiotemporal developmental progression was observed morphologically and functionally with basal cochlea maturation preceding apical cochlea by 2-3 d in all measured properties. The fraction of mechanosensitive cells increased rapidly, with a midpoint at postnatal day 0 for basal cells, and correlated with myosin IIIa immunoreactivity. MET current magnitude increased over several days. Adaptation lagged the onset of transduction by a day and matured more slowly, overlapping but preceding the rise in myosin Ic immunoreactivity. Less than approximately 25% of myosin Ic expression was required for the mature adaptation response, suggesting multiple roles for this protein in hair bundle function. Directional sensitivity, lacking in immature responses, developed rapidly and correlated with the pruning of radial links and an increase in tenting of stereociliary tips. Morphological and electrophysiological data support a hypothesis in which key elements arrive independently at the site of MET, with a mature response occurring as membrane tension increases, likely by the increased tensioning of the tip link with the onset of adaptation. Organotypic cultures developed normal, tonotopically specific, MET response properties, suggesting that maturation was not influenced significantly by external factors such as innervation, endolymph, normal mechanical stimulation, or an intact organ of Corti.

A new compartment at stereocilia tips defined by spatial and temporal patterns of myosin IIIa expression.

Class III myosins are motor proteins that contain an N-terminal kinase domain and a C-terminal actin-binding domain. We show that myosin IIIa, which has been implicated in nonsyndromic progressive hearing loss, is localized at stereocilia tips. Myosin IIIa progressively accumulates during stereocilia maturation in a thimble-like pattern around the stereocilia tip, distinct from the cap-like localization of myosin XVa and the shaft localization of myosin Ic. Overexpression of deletion mutants for functional domains of green fluorescent protein (GFP)-myosin IIIa shows that the motor domain, but not the actin-binding tail domain, is required for stereocilia tip localization. Deletion of the kinase domain produces stereocilia elongation and bulging of the stereocilia tips. The thimble-like localization and the influence myosin IIIa has on stereocilia shape reveal a previously unrecognized molecular compartment at the distal end of stereocilia, the site of actin polymerization as well as operation of the mechanoelectrical transduction apparatus.

Corrigendum: Stereocilia-staircase spacing is influenced by myosin III motors and their cargos espin-1 and espin-like.

This corrects the article DOI: 10.1038/ncomms10833.

Stereocilia-staircase spacing is influenced by myosin III motors and their cargos espin-1 and espin-like.

Hair cells tightly control the dimensions of their stereocilia, which are actin-rich protrusions with graded heights that mediate mechanotransduction in the inner ear. Two members of the myosin-III family, MYO3A and MYO3B, are thought to regulate stereocilia length by transporting cargos that control actin polymerization at stereocilia tips. We show that eliminating espin-1 (ESPN-1), an isoform of ESPN and a myosin-III cargo, dramatically alters the slope of the stereocilia staircase in a subset of hair cells. Furthermore, we show that espin-like (ESPNL), primarily present in developing stereocilia, is also a myosin-III cargo and is essential for normal hearing. ESPN-1 and ESPNL each bind MYO3A and MYO3B, but differentially influence how the two motors function. Consequently, functional properties of different motor-cargo combinations differentially affect molecular transport and the length of actin protrusions. This mechanism is used by hair cells to establish the required range of stereocilia lengths within a single cell.

CLIC5 stabilizes membrane-actin filament linkages at the base of hair cell stereocilia in a molecular complex with radixin, taperin, and myosin VI.

Chloride intracellular channel 5 protein (CLIC5) was originally isolated from microvilli in complex with actin binding proteins including ezrin, a member of the Ezrin-Radixin-Moesin (ERM) family of membrane-cytoskeletal linkers. CLIC5 concentrates at the base of hair cell stereocilia and is required for normal hearing and balance in mice, but its functional significance is poorly understood. This study investigated the role of CLIC5 in postnatal development and maintenance of hair bundles. Confocal and scanning electron microscopy of CLIC5-deficient jitterbug (jbg) mice revealed progressive fusion of stereocilia as early as postnatal day 10. Radixin (RDX), protein tyrosine phosphatase receptor Q (PTPRQ), and taperin (TPRN), deafness-associated proteins that also concentrate at the base of stereocilia, were mislocalized in fused stereocilia of jbg mice. TPRQ and RDX were dispersed even prior to stereocilia fusion. Biochemical assays showed interaction of CLIC5 with ERM proteins, TPRN, and possibly myosin VI (MYO6). In addition, CLIC5 and RDX failed to localize normally in fused stereocilia of MYO6 mutant mice. Based on these findings, we propose a model in which these proteins work together as a complex to stabilize linkages between the plasma membrane and subjacent actin cytoskeleton at the base of stereocilia.

Mechanisms of hearing loss after blast injury to the ear.

Given the frequent use of improvised explosive devices (IEDs) around the world, the study of traumatic blast injuries is of increasing interest. The ear is the most common organ affected by blast injury because it is the body's most sensitive pressure transducer. We fabricated a blast chamber to re-create blast profiles similar to that of IEDs and used it to develop a reproducible mouse model to study blast-induced hearing loss. The tympanic membrane was perforated in all mice after blast exposure and found to heal spontaneously. Micro-computed tomography demonstrated no evidence for middle ear or otic capsule injuries; however, the healed tympanic membrane was thickened. Auditory brainstem response and distortion product otoacoustic emission threshold shifts were found to be correlated with blast intensity. As well, these threshold shifts were larger than those found in control mice that underwent surgical perforation of their tympanic membranes, indicating cochlear trauma. Histological studies one week and three months after the blast demonstrated no disruption or damage to the intra-cochlear membranes. However, there was loss of outer hair cells (OHCs) within the basal turn of the cochlea and decreased spiral ganglion neurons (SGNs) and afferent nerve synapses. Using our mouse model that recapitulates human IED exposure, our results identify that the mechanisms underlying blast-induced hearing loss does not include gross membranous rupture as is commonly believed. Instead, there is both OHC and SGN loss that produce auditory dysfunction.

Myosin IIIB uses an actin-binding motif in its espin-1 cargo to reach the tips of actin protrusions.

Myosin IIIA (MYO3A) targets actin protrusion tips using a motility mechanism dependent on both motor and tail actin-binding activity [1]. We show that myosin IIIB (MYO3B) lacks tail actin-binding activity and is unable to target COS7 cell filopodia tips, yet is somehow able to target stereocilia tips. Strikingly, when MYO3B is coexpressed with espin-1 (ESPN1), a MYO3A cargo protein endogenously expressed in stereocilia [2], MYO3B targets and carries ESPN1 to COS7 filopodia tips. We show that this tip localization is lost when we remove the ESPN1 C terminus actin-binding site. We also demonstrate that, like MYO3A [2], MYO3B can elongate filopodia by transporting ESPN1 to the polymerizing end of actin filaments. The mutual dependence of MYO3B and ESPN1 for tip localization reveals a novel mechanism for the cell to regulate myosin tip localization via a reciprocal relationship with cargo that directly participates in actin binding for motility. Our results are consistent with a novel form of motility for class III myosins that requires both motor and tail domain actin-binding activity and show that the actin-binding tail can be replaced by actin-binding cargo. This study also provides a framework to better understand the late-onset hearing loss phenotype in patients with MYO3A mutations.

Integrating the biophysical and molecular mechanisms of auditory hair cell mechanotransduction.

Mechanosensation is a primitive and somewhat ubiquitous sense. At the inner ear, sensory hair cells are refined to enhance sensitivity, dynamic range and frequency selectivity. Thirty years ago, mechanisms of mechanotransduction and adaptation were well accounted for by simple mechanical models that incorporated physiological and morphological properties of hair cells. Molecular and genetic tools, coupled with new optical techniques, are now identifying and localizing specific components of the mechanotransduction machinery. These new findings challenge long-standing theories, and require modification of old and development of new models. Future advances require the integration of molecular and physiological data to causally test these new hypotheses.

Myosin IIIa boosts elongation of stereocilia by transporting espin 1 to the plus ends of actin filaments.

Two proteins implicated in inherited deafness, myosin IIIa, a plus-end-directed motor, and espin, an actin-bundling protein containing the actin-monomer-binding motif WH2, have been shown to influence the length of mechanosensory stereocilia. Here we report that espin 1, an ankyrin repeat-containing isoform of espin, colocalizes with myosin IIIa at stereocilia tips and interacts with a unique conserved domain of myosin IIIa. We show that combined overexpression of these proteins causes greater elongation of stereocilia, compared with overexpression of either myosin IIIa alone or espin 1 alone. When these two proteins were co-expressed in the fibroblast-like COS-7 cell line they induced a tenfold elongation of filopodia. This extraordinary filopodia elongation results from the transport of espin 1 to the plus ends of F-actin by myosin IIIa and depends on espin 1 WH2 activity. This study provides the basis for understanding the role of myosin IIIa and espin 1 in regulating stereocilia length, and presents a physiological example where myosins can boost elongation of actin protrusions by transporting actin regulatory factors to the plus ends of actin filaments.

New source of muscle-derived stem cells with potential for alveolar bone reconstruction in cleft lip and/or palate patients.

Cleft lip and palate (CLP), one of the most frequent congenital malformations, affects the alveolar bone in the great majority of the cases, and the reconstruction of this defect still represents a challenge in the rehabilitation of these patients. One of the current most promising strategy to achieve this goal is the use of bone marrow stem cells (BMSC); however, isolation of BMSC or iliac bone, which is still the mostly used graft in the surgical repair of these patients, confers site morbidity to the donor. Therefore, in order to identify a new alternative source of stem cells with osteogenic potential without conferring morbidity to the donor, we have used orbicular oris muscle (OOM) fragments, which are regularly discarded during surgery repair (cheiloplasty) of CLP patients. We obtained cells from OOM fragments of four unrelated CLP patients (CLPMDSC) using previously described preplating technique. These cells, through flow cytometry analysis, were mainly positively marked for five mesenchymal stem cell antigens (CD29, CD90, CD105, SH3, and SH4), while negative for hematopoietic cell markers, CD14, CD34, CD45, and CD117, and for endothelial cell marker, CD31. After induction under appropriate cell culture conditions, these cells were capable to undergo chondrogenic, adipogenic, osteogenic, and skeletal muscle cell differentiation, as evidenced by immunohistochemistry. We also demonstrated that these cells together with a collagen membrane lead to bone tissue reconstruction in a critical-size cranial defects previously induced in nonimmunocompromised rats. The presence of human DNA in the new bone was confirmed by PCR with human-specific primers and immunohistochemistry with human nuclei antibodies. In conclusion, we showed that cells from OOM have phenotypic and behavior characteristics similar to other adult stem cells, both in vitro and in vivo. Our findings suggest that these cells represent a promising source of stem cells for alveolar bone grafting treatment, particularly in young CLP patients.