ABSTRACT: Hearing and balance rely on the transduction of mechanical stimuli arising from sound waves or head movements into electrochemical signals. This archetypal mechanoelectrical transduction process occurs in the hair-cell stereocilia of the inner ear, which experience continuous oscillations driven by undulations in the endolymph in which they are immersed. The filamentous structures called tip links, formed by an intertwined thread composed of an heterotypic complex of cadherin 23 and protocadherin 15 ectodomain dimers, connect each stereocilium to the tip of the lower sterocilium, and must maintain their integrity against continuous stimulatory deflections. By using single molecule force spectroscopy, here we demonstrate that in contrast to the case of classical cadherins, tip-link cadherins are mechanoresilient structures even at the exceptionally low Ca2+ concentration of the endolymph. We also show that the D101G deafness point mutation in cadherin 23, which affects a Ca2+ coordination site, exhibits an altered mechanical phenotype at the physiological Ca2+ concentration. Our results show a remarkable case of functional adaptation of a protein's nanomechanics to extremely low Ca2+ concentrations and pave the way to a full understanding of the mechanotransduction mechanism mediated by auditory cadherins.
Project description:Cadherin 23 and protocadherin 15 are components of tip links, fine filaments that interlink the stereocilia of hair cells and are believed to gate the hair cell's mechanotransducer channels. Tip links are aligned along the hair bundle's axis of mechanosensitivity, stretching obliquely from the top of one stereocilium to the side of an adjacent, taller stereocilium. In guinea pig auditory hair cells, tip links are polarized with cadherin 23 at the upper end and protocadherin 15 at the lower end, where the transducer channel is located. Double immunogold labeling of avian hair cells was used to study the distribution of these two proteins in kinocilial links, a link type that attaches the tallest stereocilia of the hair bundle to the kinocilium. In the kinocilial links of vestibular hair bundles, cadherin 23 localizes to the stereocilium and protocadherin 15 to the kinocilium. The two cadherins are therefore asymmetrically distributed within the kinocilial links but of a polarity that is, within those links that are aligned along the hair bundle's axis of sensitivity, reversed relative to that of tip links. Conventional transmission electron microscopy of hair bundles fixed in the presence of tannic acid reveals a distinct density in the 120-130 nm long kinocilial links that is located 35-40 nm from the kinociliary membrane. The location of this density is consistent with it being the site at which interactions occur in an in trans configuration between the opposing N-termini of homodimeric forms of cadherin 23 and protocadherin 15.
Project description:Cadherins form a large family of proteins often involved in calcium-dependent cellular adhesion. Although classical members of the family can provide a physical bond between cells, a subset of special cadherins use their extracellular domains to interlink apical specializations of single epithelial sensory cells. Two of these cadherins, cadherin-23 (CDH23) and protocadherin-15 (PCDH15), form extracellular "tip link" filaments that connect apical bundles of stereocilia on hair cells essential for inner-ear mechanotransduction. As these bundles deflect in response to mechanical stimuli from sound or head movements, tip links gate hair-cell mechanosensitive channels to initiate sensory perception. Here, we review the unusual and diverse structural properties of these tip-link cadherins and the functional significance of their deafness-related missense mutations. Based on the structural features of CDH23 and PCDH15, we discuss the elasticity of tip links and models that bridge the gap between the nanomechanics of cadherins and the micromechanics of hair-cell bundles during inner-ear mechanotransduction.
Project description:Cadherins form a large family of calcium-dependent cell-cell adhesion receptors involved in development, morphogenesis, synaptogenesis, differentiation, and carcinogenesis through signal mechanotransduction using an adaptor complex that connects them to the cytoskeleton. However, the molecular mechanisms underlying mechanotransduction through cadherins remain unknown, although their extracellular region (ectodomain) is thought to be critical in this process. By single molecule force spectroscopy, molecular dynamics simulations, and protein engineering, here we have directly examined the nanomechanics of the C-cadherin ectodomain and found it to be strongly dependent on the calcium concentration. In the presence of calcium, the ectodomain extends through a defined ("canalized") pathway that involves two mechanical resistance elements: a mechanical clamp from the cadherin domains and a novel mechanostable component from the interdomain calcium-binding regions ("calcium rivet") that is abolished by magnesium replacement and in a mutant intended to impede calcium coordination. By contrast, in the absence of calcium, the mechanical response of the ectodomain becomes largely "decanalized" and destabilized. The cadherin ectodomain may therefore behave as a calcium-switched "mechanical antenna" with very different mechanical responses depending on calcium concentration (which would affect its mechanical integrity and force transmission capability). The versatile mechanical design of the cadherin ectodomain and its dependence on extracellular calcium facilitate a variety of mechanical responses that, we hypothesize, could influence the various adhesive properties mediated by cadherins in tissue morphogenesis, synaptic plasticity, and disease. Our work represents the first step toward the mechanical characterization of the cadherin system, opening the door to understanding the mechanical bases of its mechanotransduction.
Project description: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.
Project description:The hair-cell tip link, a fine filament directly conveying force to mechanosensitive transduction channels, is composed of two proteins, protocadherin-15 and cadherin-23, whose mutation causes deafness. However, their molecular structure, elasticity, and deafness-related structural defects are unknown. We present crystal structures of the first and second extracellular cadherin repeats of cadherin-23. Overall, structures show typical cadherin folds, but reveal an elongated N terminus that precludes classical cadherin interactions and contributes to an N-terminal Ca(2+)-binding site. The deafness mutation D101G, in the linker region between the repeats, causes a slight bend between repeats and decreases Ca(2+) affinity. Molecular dynamics simulations suggest that cadherin-23 repeats are stiff and that either removing Ca(2+) or mutating Ca(2+)-binding residues reduces rigidity and unfolding strength. The structures define an uncharacterized cadherin family and, with simulations, suggest mechanisms underlying inherited deafness and how cadherin-23 may bind with itself and with protocadherin-15 to form the tip link.
Project description:Hearing and balance use hair cells in the inner ear to transform mechanical stimuli into electrical signals. Mechanical force from sound waves or head movements is conveyed to hair-cell transduction channels by tip links, fine filaments formed by two atypical cadherins known as protocadherin 15 and cadherin 23 (refs 4, 5). These two proteins are involved in inherited deafness and feature long extracellular domains that interact tip-to-tip in a Ca(2+)-dependent manner. However, the molecular architecture of this complex is unknown. Here we combine crystallography, molecular dynamics simulations and binding experiments to characterize the protocadherin 15-cadherin 23 bond. We find a unique cadherin interaction mechanism, in which the two most amino-terminal cadherin repeats (extracellular cadherin repeats 1 and 2) of each protein interact to form an overlapped, antiparallel heterodimer. Simulations predict that this tip-link bond is mechanically strong enough to resist forces in hair cells. In addition, the complex is shown to become unstable in response to Ca(2+) removal owing to increased flexure of Ca(2+)-free cadherin repeats. Finally, we use structures and biochemical measurements to study the molecular mechanisms by which deafness mutations disrupt tip-link function. Overall, our results shed light on the molecular mechanics of hair-cell sensory transduction and on new interaction mechanisms for cadherins, a large protein family implicated in tissue and organ morphogenesis, neural connectivity and cancer.
Project description:Tight regulation of calcium (Ca2+) concentrations in the stereocilia bundles of auditory hair cells of the inner ear is critical to normal auditory transduction. The plasma membrane Ca2+ ATPase 2 (PMCA2), encoded by the Atp2b2 gene, is the primary mechanism for clearance of Ca2+ from auditory stereocilia, keeping intracellular levels low, and also contributes to maintaining adequate levels of extracellular Ca2+ in the endolymph. This study characterizes a novel null Atp2b2 allele, dfw(i5), by examining cochlear anatomy, vestibular function and auditory physiology in mutant mice. Loss of auditory function in PMCA2 mutants can be attributed to dysregulation of intracellular Ca2+ inside the stereocilia bundles. However, extracellular Ca2+ ions surrounding the stereocilia are also required for rigidity of cadherin 23, a component of the stereocilia tip-link encoded by the Cdh23 gene. This study further resolves the interaction between Atp2b2 and Cdh23 in a gene dosage and frequency-dependent manner, and finds that low frequencies are significantly affected by the interaction. In +/dfw(i5) mice, one mutant copy of Cdh23 is sufficient to cause broad frequency hearing impairment. Additionally, we report another modifying interaction with Atp2b2 on auditory sensitivity, possibly caused by an unidentified hearing loss gene in mice.
Project description:Tip links are thought to gate the mechanically sensitive transduction channels of hair cells, but how they form during development and regeneration remains mysterious. In particular, it is unclear how tip links are strung between stereocilia so that they are oriented parallel to a single axis; why their polarity is uniform despite their constituent molecules' intrinsic asymmetry; and why only a single tip link is present at each tip-link position. We present here a series of simple rules that reasonably explain why these phenomena occur. In particular, our model relies on each of the two ends of the tip link having distinct Ca2+-dependent stability and being connected to different motor complexes. A simulation employing these rules allowed us to explore the parameter space for the model, demonstrating the importance of the feedback between transduction channels and angled links, links that are 60° off-axis with respect to mature tip links. We tested this key aspect of the model by examining angled links in chick cochlea hair cells. As implied by the assumptions used to generate the model, we found that angled links were stabilized if there was no tip link at the tip of the upper stereocilium, and appeared when transduction channels were blocked. The model thus plausibly explains how tip-link formation and pruning can occur.
Project description:Ca2+ enters the stereocilia of hair cells through mechanoelectrical transduction channels opened by the deflection of the hair bundle and is exported back to endolymph by an unusual splicing isoform (w/a) of plasma-membrane calcium-pump isoform 2 (PMCA2). Ablation or missense mutations of the pump cause deafness, as described for the G283S mutation in the deafwaddler (dfw) mouse. A deafness-inducing missense mutation of PMCA2 (G293S) has been identified in a human family. The family also was screened for mutations in cadherin 23, which accentuated hearing loss in a previously described human family with a PMCA2 mutation. A T1999S substitution was detected in the cadherin 23 gene of the healthy father and affected son but not in that of the unaffected mother, who presented instead the PMCA2 mutation. The w/a isoform was overexpressed in CHO cells. At variance with the other PMCA2 isoforms, it became activated only marginally when exposed to a Ca2+ pulse. The G293S and G283S mutations delayed the dissipation of Ca2+ transients induced in CHO cells by InsP3. In organotypic cultures, Ca2+ imaging of vestibular hair cells showed that the dissipation of stereociliary Ca2+ transients induced by Ca2+ uncaging was compromised in the dfw and PMCA2 knockout mice, as was the sensitivity of the mechanoelectrical transduction channels to hair bundle displacement in cochlear hair cells.
Project description:The 6-billion human population provides a vast reservoir of mutations, which, in addition to the opportunity of detecting very subtle defects, including specific cognitive dysfunctions as well as late appearing disorders, offers a unique background in which to investigate the roles of cell-cell adhesion proteins. Here we focus on inherited human disorders involving members of the cadherin superfamily. Most of the advances concern monogenic disorders. Yet, with the development of single nucleotide polymorphism (SNP) association studies, cadherin genes are emerging as susceptibility genes in multifactorial disorders. Various skin and heart disorders revealed the critical role played by desmosomal cadherins in epidermis, hairs, and myocardium, which experience high mechanical stress. Of particular interest in that respect is the study of Usher syndrome type 1 (USH1), a hereditary syndromic form of deafness. Studies of USH1 brought to light the crucial role of transient fibrous links formed by cadherin 23 and protocadherin 15 in the cohesion of the developing hair bundle, the mechanoreceptive structure of the auditory sensory cells, as well as the involvement of these cadherins in the formation of the tip-link, a key component of the mechano-electrical transduction machinery. Finally, in line with the well-established role of cadherins in synaptic formation, maintenance, strength, and plasticity, a growing number of cadherin family members, especially protocadherins, have been found to be involved in neuropsychiatric disorders.