Rab11a Is Essential for the Development and Integrity of the Stereocilia and Kinocilia in the Mammalian Organ of Corti.

The cochlea hair cells transform mechanic sounds to neural signals with a remarkable sensitivity and resolution. This is achieved via the precisely sculpted mechanotransduction apparatus of the hair cells and the supporting structure of the cochlea. The shaping of the mechanotransduction apparatus, the staircased stereocilia bundles on the apical surface of the hair cells, requires an intricate regulatory network including planar cell polarity (PCP) and primary cilia genes in orienting stereocilia bundles and building molecular machinery of the apical protrusions. The mechanism linking these regulatory components is unknown. Here, we show that a small GTPase known for its role in protein trafficking, Rab11a, is required for ciliogenesis in hair cells during development in mice. In addition, in the absence of Rab11a, stereocilia bundles lost their cohesion and integrity, and mice are deaf. These data indicate an essential role of protein trafficking in the formation of hair cell mechanotransduction apparatus, implicating a role of Rab11a or protein trafficking in linking the cilia and polarity regulatory components with the molecular machinery in building the cohesive and precisely shaped stereocilia bundles.

Interpreting the Evolutionary Echoes of a Protein Complex Essential for Inner-Ear Mechanosensation.

The sensory epithelium of the inner ear, found in all extant lineages of vertebrates, has been subjected to over 500 million years of evolution, resulting in the complex inner ear of modern vertebrates. Inner-ear adaptations are as diverse as the species in which they are found, and such unique anatomical variations have been well studied. However, the evolutionary details of the molecular machinery that is required for hearing are less well known. Two molecules that are essential for hearing in vertebrates are cadherin-23 and protocadherin-15, proteins whose interaction with one another acts as the focal point of force transmission when converting sound waves into electrical signals that the brain can interpret. This "tip-link" interaction exists in every lineage of vertebrates, but little is known about the structure or mechanical properties of these proteins in most non-mammalian lineages. Here, we use various techniques to characterize the evolution of this protein interaction. Results show how evolutionary sequence changes in this complex affect its biophysical properties both in simulations and experiments, with variations in interaction strength and dynamics among extant vertebrate lineages. Evolutionary simulations also characterize how the biophysical properties of the complex in turn constrain its evolution and provide a possible explanation for the increase in deafness-causing mutants observed in cadherin-23 relative to protocadherin-15. Together, these results suggest a general picture of tip-link evolution in which selection acted to modify the tip-link interface, although subsequent neutral evolution combined with varying degrees of purifying selection drove additional diversification in modern tetrapods.

The tetraspan LHFPL5 is critical to establish maximal force sensitivity of the mechanotransduction channel of cochlear hair cells.

The mechanoelectrical transduction (MET) channel of cochlear hair cells is gated by the tip link, but the mechanisms that establish the exquisite force sensitivity of this MET channel are not known. Here, we show that the tetraspan lipoma HMGIC fusion partner-like 5 (LHFPL5) directly couples the tip link to the MET channel. Disruption of these interactions severely perturbs MET. Notably, the N-terminal cytoplasmic domain of LHFPL5 binds to an amphipathic helix in TMC1, a critical gating domain conserved between different MET channels. Mutations in the amphipathic helix of TMC1 or in the N-terminus of LHFPL5 that perturb interactions of LHFPL5 with the amphipathic helix affect channel responses to mechanical force. We conclude that LHFPL5 couples the tip link to the MET channel and that channel gating depends on a structural element in TMC1 that is evolutionarily conserved between MET channels. Overall, our findings support a tether model for transduction channel gating by the tip link.

Template-independent genome editing in the Pcdh15av-3j mouse, a model of human DFNB23 nonsyndromic deafness.

Although frameshift mutations lead to 22% of inherited Mendelian disorders in humans, there is no efficient in vivo gene therapy strategy available to date, particularly in nondividing cells. Here, we show that nonhomologous end-joining (NHEJ)-mediated nonrandom editing profiles compensate the frameshift mutation in the Pcdh15 gene and restore the lost mechanotransduction function in postmitotic hair cells of Pcdh15av-3J mice, an animal model of human nonsyndromic deafness DFNB23. Identified by an ex vivo evaluation system in cultured cochlear explants, the selected guide RNA restores reading frame in approximately 50% of indel products and recovers mechanotransduction in more than 70% of targeted hair cells. In vivo treatment shows that half of the animals gain improvements in auditory responses, and balance function is restored in the majority of injected mutant mice. These results demonstrate that NHEJ-mediated reading-frame restoration is a simple and efficient strategy in postmitotic systems.

Putting the Pieces Together: the Hair Cell Transduction Complex.

Identification of the components of the mechanosensory transduction complex in hair cells has been a major research interest for many auditory and vestibular scientists and has attracted attention from outside the field. The past two decades have witnessed a number of significant advances with emergence of compelling evidence implicating at least a dozen distinct molecular components of the transduction machinery. Yet, how the pieces of this ensemble fit together and function in harmony to enable the senses of hearing and balance has not been clarified. The goal of this review is to summarize a 2021 symposium presented at the annual mid-winter meeting of the Association for Research in Otolaryngology. The symposium brought together the latest insights from within and beyond the field to examine individual components of the transduction complex and how these elements interact at molecular, structural, and biophysical levels to gate mechanosensitive channels and initiate sensory transduction in the inner ear. The review includes a brief historical background to set the stage for topics to follow that focus on structure, properties, and interactions of proteins such as CDH23, PCDH15, LHFPL5, TMIE, TMC1/2, and CIB2/3. We aim to present the diversity of ideas in this field and highlight emerging theories and concepts. This review will not only provide readers with a deeper appreciation of the components of the transduction apparatus and how they function together, but also bring to light areas of broad agreement, areas of scientific controversy, and opportunities for future scientific discovery.

Weakening of interaction networks with aging in tip-link protein induces hearing loss.

Age-related hearing loss (ARHL) is a common condition in humans marking the gradual decrease in hearing with age. Perturbations in the tip-link protein cadherin-23 that absorbs the mechanical tension from sound and maintains the integrity of hearing is associated with ARHL. Here, in search of molecular origins for ARHL, we dissect the conformational behavior of cadherin-23 along with the mutant S47P that progresses the hearing loss drastically. Using an array of experimental and computational approaches, we highlight a lower thermodynamic stability, significant weakening in the hydrogen-bond network and inter-residue correlations among ß-strands, due to the S47P mutation. The loss in correlated motions translates to not only a remarkable two orders of magnitude slower folding in the mutant but also to a proportionately complex unfolding mechanism. We thus propose that loss in correlated motions within cadherin-23 with aging may trigger ARHL, a molecular feature that likely holds true for other disease-mutations in ß-strand-rich proteins.

Structural determinants of protocadherin-15 mechanics and function in hearing and balance perception.

The vertebrate inner ear, responsible for hearing and balance, is able to sense minute mechanical stimuli originating from an extraordinarily broad range of sound frequencies and intensities or from head movements. Integral to these processes is the tip-link protein complex, which conveys force to open the inner-ear transduction channels that mediate sensory perception. Protocadherin-15 and cadherin-23, two atypically large cadherins with 11 and 27 extracellular cadherin (EC) repeats, are involved in deafness and balance disorders and assemble as parallel homodimers that interact to form the tip link. Here we report the X-ray crystal structure of a protocadherin-15 + cadherin-23 heterotetrameric complex at 2.9-Å resolution, depicting a parallel homodimer of protocadherin-15 EC1-3 molecules forming an antiparallel complex with two cadherin-23 EC1-2 molecules. In addition, we report structures for 10 protocadherin-15 fragments used to build complete high-resolution models of the monomeric protocadherin-15 ectodomain. Molecular dynamics simulations and validated crystal contacts are used to propose models for the complete extracellular protocadherin-15 parallel homodimer and the tip-link bond. Steered molecular dynamics simulations of these models suggest conditions in which a structurally diverse and multimodal protocadherin-15 ectodomain can act as a stiff or soft gating spring. These results reveal the structural determinants of tip-link-mediated inner-ear sensory perception and elucidate protocadherin-15's structural and adhesive properties relevant in disease.

Myosin VII, USH1C, and ANKS4B or USH1G Together Form Condensed Molecular Assembly via Liquid-Liquid Phase Separation.

Hair cell stereocilia tip-links function to sense mechanical forces generated by sound waves and maintain the structure of stereocilia by rooting the tail of cadherins to highly dense structures known as tip-link densities. Although the molecular components are largely known, the mechanisms underlying the tip-link density formation are unknown. Here, we show that Myosin VIIB (MYO7B), USH1C, and ANKS4B, which form a specific complex stabilizing tip-links in intestine microvilli, could form dense condensates via liquid-liquid phase separation in vitro and in cells. The MYO7A, USH1C, and USH1G complex also undergoes phase separation in cells. Formation of the MYO7A/USH1C/USH1G and MYO7B/USH1C/ANKS4B condensates requires strong and multivalent interactions between proteins in both tripartite complexes. Point mutations of MYO7A found in Usher syndrome patients weaken or even disrupt the multivalent interactions of the MYO7A/USH1C/USH1G complex and impair its phase separation. Thus, the stereocilia tip-link densities may form via phase separation of the MYO7A/USH1C/USH1G complex.

Mechanisms of Hair Cell Damage and Repair.

Sensory hair cells of the inner ear are exposed to continuous mechanical stress, causing damage over time. The maintenance of hair cells is further challenged by damage from a variety of other ototoxic factors, including loud noise, aging, genetic defects, and ototoxic drugs. This damage can manifest in many forms, from dysfunction of the hair cell mechanotransduction complex to loss of specialized ribbon synapses, and may even result in hair cell death. Given that mammalian hair cells do not regenerate, the repair of hair cell damage is important for continued auditory function throughout life. Here, we discuss how several key hair cell structures can be damaged, and what is known about how they are repaired.

Localization of TMC1 and LHFPL5 in auditory hair cells in neonatal and adult mice.

The channel that governs mechanotransduction (MT) by hair cells in the inner ear has been investigated intensively for 4 decades, but its precise molecular composition remains enigmatic. Transmembrane channel-like protein 1 (TMC1) was recently identified as a component of the MT channel, and lipoma HMGIC fusion partner-like 5 (LHFPL5) is considered to be part of the MT complex and may functionally couple the tip link to the MT channel. As components of the MT complex, TMC1 and LHFPL5 are expected to localize at the lower end of the tip link in hair cells, a notion generally supported by previous studies on neonatal mice. However, the localization of these 2 proteins, particularly in the hair cells of adult mice, remains incompletely elucidated. Because determination of TMC1 and LHFPL5 localization at distinct developmental stages is essential for understanding their function and regulation, we used several approaches to examine the localization of these proteins in neonatal and adult hair cells in the mouse. We report several notable findings: 1) TMC1 and LHFPL5 predominantly localize at the tip of the shorter rows of stereocilia in neonatal hair cells, which largely verifies the previously published findings in neonatal hair cells; 2) LHFPL5 persists in the hair bundle of hair cells after postnatal day (P)7, which clarifies the previously reported unexpected absence of LHFPL5 after P7 and supports the view that LHFPL5 is a permanent component in the MT complex; and 3) TMC1 and LHFPL5 remain at the tip of the shorter rows of stereocilia in adult outer hair cells, but in adult inner hair cells, TMC1 is uniformly distributed in both the tallest row and the shorter rows of stereocilia, whereas LHFPL5 is uniformly distributed in the shorter rows of stereocilia. These findings raise intriguing questions regarding the turnover rate, regulation, additional functions, and functional interaction of TMC1 and LHFPL5. Our study confirms the previous findings in neonatal hair cells and reveals several previously unidentified aspects of TMC1 and LHFPL5 localization in more mature hair cells.-Li, X., Yu, X., Chen, X., Liu, Z., Wang, G., Li, C., Wong, E. Y. M., Sham, M. H., Tang, J., He, J., Xiong, W., Liu, Z., Huang, P. Localization of TMC1 and LHFPL5 in auditory hair cells in neonatal and adult mice.

Zooming in on Cadherin-23: Structural Diversity and Potential Mechanisms of Inherited Deafness.

Cadherin-23 (CDH23) is an essential component of hair-cell tip links, fine filaments that mediate inner-ear mechanotransduction. The extracellular domain of CDH23 forms about three-fourths of the tip link with 27 extracellular cadherin (EC) repeats that are structurally similar but not identical to each other. Calcium (Ca2+) coordination at the EC linker regions is key for tip-link elasticity and function. There are ∼116 sites in CDH23 affected by deafness-causing mutations, many of which alter conserved Ca2+-binding residues. Here we present crystal structures showing 18 CDH23 EC repeats, including the most and least conserved, a fragment carrying disease mutations, and EC repeats with non-canonical Ca2+-binding motif sequences and unusual secondary structure. Complementary experiments show deafness mutations' effects on stability and affinity for Ca2+. Additionally, a model of nine contiguous CDH23 EC repeats reveals helicity and potential parallel dimerization faces. Overall, our studies provide detailed structural insight into CDH23 function in mechanotransduction.

Mechanotransduction by PCDH15 Relies on a Novel cis-Dimeric Architecture.

The tip link, a filament formed by protocadherin 15 (PCDH15) and cadherin 23, conveys mechanical force from sound waves and head movement to open hair-cell mechanotransduction channels. Tip-link cadherins are thought to have acquired structural features critical for their role in mechanotransduction. Here, we biophysically and structurally characterize the unusual cis-homodimeric architecture of PCDH15. We show that PCDH15 molecules form double-helical assemblies through cis-dimerization interfaces in the extracellular cadherin EC2-EC3 domain region and in a unique membrane-proximal domain. Electron microscopy studies visualize the cis-dimeric PCDH15 assembly and reveal the PCDH15 extracellular domain as a parallel double helix with cis cross-bridges at the two locations we defined. The helical configuration suggests the potential for elasticity through helix winding and unwinding. Functional studies in hair cells show that mutations that perturb PCDH15 dimerization contacts affect mechanotransduction. Together, these data reveal the cis-dimeric architecture of PCDH15 and show that dimerization is critical for sensing mechanical stimuli.

Recombinant protein of the first two ectodomains of cadherin 23 from erl mice shows impairment in Ca2+-dependent proteolysis protection.

The erl mouse is a mouse model of nonsyndromic autosomal recessive deafness (DFNB12) on the C57BL/6J background. This project was carried out to express the first two ectodomains of cadherin 23 (CDH23 EC1+2) of erl mice in Escherichia coli and identify the Ca2+-binding ability of the recombinant protein. DNA sequences of CDH23 EC1+2 from wild type and erl mice were synthesized and cloned into pBV220 plasmids. Recombinant plasmids were transformed into Escherichia coli and expression of CDH23 EC1+2 was induced by increasing the temperature from 30 °C to 42 °C. The proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and antigenicity of proteins was identified by Western Blotting. Inclusion bodies were denatured in 8 M urea, purified by ion-exchange and gel filtration chromatography and refolded with dialysis in buffer containing 0.1% sarkosyl. The Ca2+-binding ability of CDH23 EC1+2 was determined by Ca2+-dependent proteolysis protection. The results showed that the sizes and sequences of inserts in recombinant plasmids were consistent with expectation and that the recombinant proteins were found mainly in the form of inclusion bodies which maintain antigenicity. After refolding, the secondary structures of recombinant proteins were measured by circular dichroism (CD) spectra. Moreover, CDH23 EC1+2 from the erl mice showed less Ca2+-dependent proteolysis protection comparing with that of the wild type control. We therefore concluded that impairment of Ca2+-dependent protein interaction was likely involved in the progressive hearing loss in erl mice. The results may aid in understanding the mechanism of hearing loss in DFNB12.

A Partial Calcium-Free Linker Confers Flexibility to Inner-Ear Protocadherin-15.

Tip links of the inner ear are protein filaments essential for hearing and balance. Two atypical cadherins, cadherin-23 and protocadherin-15, interact in a Ca2+-dependent manner to form tip links. The largely unknown structure and mechanics of these proteins are integral to understanding how tip links pull on ion channels to initiate sensory perception. Protocadherin-15 has 11 extracellular cadherin (EC) repeats. Its EC3-4 linker lacks several of the canonical Ca2+-binding residues, and contains an aspartate-to-alanine polymorphism (D414A) under positive selection in East Asian populations. We present structures of protocadherin-15 EC3-5 featuring two Ca2+-binding linker regions: canonical EC4-5 linker binding three Ca2+ ions, and non-canonical EC3-4 linker binding only two Ca2+ ions. Our structures and biochemical assays reveal little difference between the D414 and D414A variants. Simulations predict that the partial Ca2+-free EC3-4 linker exhibits increased flexural flexibility without compromised mechanical strength, providing insight into the dynamics of tip links and other atypical cadherins.

Molecular Identity of the Mechanotransduction Channel in Hair Cells: Not Quiet There Yet.

Hair cells in the mammalian cochlea are specialized mechanosensory cells that convert sound-induced vibrations into electrochemical signals. The molecular composition of the mechanotransduction channel underlying auditory perception has been difficult to define. The study of genes that are linked to inherited forms of deafness has recently provided tantalizing clues. Current findings indicate that the mechanotransduction channel in hair cells is a complex molecular machine. Four different proteins (TMHS/LHFPL5, TMIE, TMC1, and TMC2) have so far been linked to the transduction channel, but which proteins contribute to the channel pore still needs to be determined. Current evidence also suggests that the channel complex may contain additional, yet to be identified components.

The tip-link molecular complex of the auditory mechano-electrical transduction machinery.

Sound waves are converted into electrical signals by a process of mechano-electrical transduction (MET), which takes place in the hair bundle of cochlear hair cells. In response to the mechanical stimulus of the hair bundle, the tip-links, key components of the MET machinery, are tensioned and the MET channels open, which results in the generation of the cell receptor potential. Tip-links are composed of cadherin-23 (Cdh23) and protocadherin-15 (Pcdh15), both non-conventional cadherins, that form the upper and the lower part of these links, respectively. Here, we review the various Pcdh15 isoforms present in the organ of Corti, their localization in the auditory hair bundles, their involvement in the molecular complex forming the tip-link, and their interactions with transmembrane molecules that are components of the lower MET machinery.

The CD2 isoform of protocadherin-15 is an essential component of the tip-link complex in mature auditory hair cells.

Protocadherin-15 (Pcdh15) is a component of the tip-links, the extracellular filaments that gate hair cell mechano-electrical transduction channels in the inner ear. There are three Pcdh15 splice isoforms (CD1, CD2 and CD3), which only differ by their cytoplasmic domains; they are thought to function redundantly in mechano-electrical transduction during hair-bundle development, but whether any of these isoforms composes the tip-link in mature hair cells remains unknown. By immunolabelling and both morphological and electrophysiological analyses of post-natal hair cell-specific conditional knockout mice (Pcdh15ex38-fl/ex38-fl Myo15-cre+/-) that lose only this isoform after normal hair-bundle development, we show that Pcdh15-CD2 is an essential component of tip-links in mature auditory hair cells. The finding, in the homozygous or compound heterozygous state, of a PCDH15 frameshift mutation (p.P1515Tfs*4) that affects only Pcdh15-CD2, in profoundly deaf children from two unrelated families, extends this conclusion to humans. These results provide key information for identification of new components of the mature auditory mechano-electrical transduction machinery. This will also serve as a basis for the development of gene therapy for deafness caused by PCDH15 defects.

Usher protein functions in hair cells and photoreceptors.

The 10 different genes associated with the deaf/blind disorder, Usher syndrome, encode a number of structurally and functionally distinct proteins, most expressed as multiple isoforms/protein variants. Functional characterization of these proteins suggests a role in stereocilia development in cochlear hair cells, likely owing to adhesive interactions in hair bundles. In mature hair cells, homodimers of the Usher cadherins, cadherin 23 and protocadherin 15, interact to form a structural fiber, the tip link, and the linkages that anchor the taller stereocilia's actin cytoskeleton core to the shorter adjacent stereocilia and the elusive mechanotransduction channels, explaining the deafness phenotype when these molecular interactions are perturbed. The conundrum is that photoreceptors lack a synonymous mechanotransduction apparatus, and so a common theory for Usher protein function in the two neurosensory cell types affected in Usher syndrome is lacking. Recent evidence linking photoreceptor cell dysfunction in the shaker 1 mouse model for Usher syndrome to light-induced protein translocation defects, combined with localization of an Usher protein interactome at the periciliary region of the photoreceptors suggests Usher proteins might regulate protein trafficking between the inner and outer segments of photoreceptors. A distinct Usher protein complex is trafficked to the ribbon synapses of hair cells, and synaptic defects have been reported in Usher mutants in both hair cells and photoreceptors. This review aims to clarify what is known about Usher protein function at the synaptic and apical poles of hair cells and photoreceptors and the prospects for identifying a unifying pathobiological mechanism to explain deaf/blindness in Usher syndrome.