Cis-clustering of cadherin-23 controls the kinetics of cell-cell adhesion

Cis and trans-interactions in cadherins are the foundations of multicellularity. While the trans-interaction mediate cell-cell adhesion, the cis-interaction is postulated as strengthening to trans by clustering. The well-accepted model in cadherin-adhesion is that the trans precedes cis via a diffusion-trap kinetic model. Here we report that cadherin-23, a non-classical cadherin with an extended extracellular region, undergoes clustering in solution via lateral interactions independent of trans and phase separate as liquid droplets. In cellulo using fluorescence-recovery after the photobleaching, we noticed a significantly slow-diffusion of cadherin-23 at the intercellular junctions, indicating the diffusion of a cluster. The cis-clustering accelerates the cell-cell adhesion and, thus, kinetically controls cell-adhesion via cis precedes trans model. Though the connection of cis-clustering with the rapid adhesion is yet to explore, M2-macrophages that predominantly express cadherin-23 undergo fast attachments to circulatory tumor cells during metastasis.

Molecular structures and conformations of protocadherin-15 and its complexes on stereocilia elucidated by cryo-electron tomography

Mechanosensory transduction (MT), the conversion of mechanical stimuli into electrical signals, underpins hearing and balance and is carried out within hair cells in the inner ear. Hair cells harbor actin-filled stereocilia, arranged in rows of descending heights, where the tips of stereocilia are connected to their taller neighbors by a filament composed of protocadherin 15 (PCDH15) and cadherin 23 (CDH23), deemed the ‘tip link’. Tension exerted on the tip link opens an ion channel at the tip of the shorter stereocilia, thus converting mechanical force into an electrical signal. While biochemical and structural studies have provided insights into the molecular composition and structure of isolated portions of the tip link, the architecture, location and conformational states of intact tip links, on stereocilia, remains unknown. Here we report in situ cryo-electron microscopy imaging of the tip link in mouse stereocilia. We observe individual PCDH15 molecules at the tip and shaft of stereocilia and determine their stoichiometry, conformational heterogeneity, and their complexes with other filamentous proteins, perhaps including CDH23. The PCDH15 complexes occur in clusters, frequently with more than one copy of PCDH15 at the tip of stereocilia, suggesting that tip links might consist of more than one copy of PCDH15 complexes and, by extension, might include multiple MT complexes.

Cochlear Implantation Outcomes in Children With CDH23 Mutations–Associated Hearing Loss

Objective Mutations in the cadherin 23 gene ( CDH23) have been reported to cause cochlear damage, but few studies have investigated the auditory and speech outcome of patients after cochlear implantation. Here, we describe the genetic, auditory, and postoperative outcomes of patients with CDH23 mutations who received cochlear implants. Study Design Retrospective case review. Setting Tertiary referral center. Methods Targeted deafness-related gene panels were sequenced in Chinese families with profound sensorineural hearing loss. The clinical features of subjects carrying potentially pathogenic CDH23 mutations were analyzed. Results Between 2017 and 2019, we identified 5 children with prelinguistically profound hearing loss at our center who harbored 6 variants of CDH23 that segregated with the disease. Of these, 4 variants were novel (c.2591G>T, c.4785G>C, c.5765A>G, and c.9280_9281insTT). All affected individuals had a loss of outer hair cell function, with an average residual hearing level of 3 to 10 dB SPL. Cochlear implantations were arranged for the patients at 11 to 36 months of age. All children made gains in their hearing, language, and speech performances 14 to 120 months after surgery. Their auditory outcomes improved during follow-up intervals. Conclusion This study revealed that children with congenital cochlear defects caused by CDH23 variants can acquire an acceptable auditory and speech outcome after cochlear implantation. Early genetic detection and prenatal counseling for rare deafness genes such as CDH23 remain a priority for the future.

Synaptic Release Potentiation at Aging Auditory Ribbon Synapses

Age-related hidden hearing loss is often described as a cochlear synaptopathy that results from a progressive degeneration of the inner hair cell (IHC) ribbon synapses. The functional changes occurring at these synapses during aging are not fully understood. Here, we characterized this aging process in IHCs of C57BL/6J mice, a strain which is known to carry a cadherin-23 mutation and experiences early hearing loss with age. These mice, while displaying a large increase in auditory brainstem thresholds due to 50% loss of IHC synaptic ribbons at middle age (postnatal day 365), paradoxically showed enhanced acoustic startle reflex suggesting a hyperacusis-like response. The auditory defect was associated with a large shrinkage of the IHCs' cell body and a drastic enlargement of their remaining presynaptic ribbons which were facing enlarged postsynaptic AMPAR clusters. Presynaptic Ca2+ microdomains and the capacity of IHCs to sustain high rates of exocytosis were largely increased, while on the contrary the expression of the fast-repolarizing BK channels, known to negatively control transmitter release, was decreased. This age-related synaptic plasticity in IHCs suggested a functional potentiation of synaptic transmission at the surviving synapses, a process that could partially compensate the decrease in synapse number and underlie hyperacusis.

Molecular structure and conformation of stereocilia tip-links elucidated by cryo-electron tomography

Mechanosensory transduction (MT), the conversion of mechanical stimuli into electrical signals, underpins hearing and balance and is carried out within hair cells in the inner ear. Hair cells harbor actin-filled stereocilia, arranged in rows of descending heights, where the tips of stereocilia are connected to their taller neighbors by a filament composed of protocadherin 15 (PCDH15) and cadherin 23 (CDH23), deemed the ‘tip-link’. Tension exerted on the tip-link opens an ion channel at the tip of the shorter stereocilia, thus converting mechanical force into an electrical signal. While biochemical and structural studies have provided insights into the molecular composition and structure of isolated portions of the tip-link, the architecture, location and conformational states of intact tip-links, on stereocilia, remains unknown. Here we report in situ cryo-electron microscopy imaging of the tip-link in mouse stereocilia. We observe individual PCDH15 molecules at the tip and shaft of stereocilia and determine their stoichiometry, conformational heterogeneity, and their complexes with CDH23. The PCDH15/CDH23 complexes occur in clusters, frequently with more than one copy of PCDH15 at the tip of stereocilia, suggesting that tip-links might consist of more than one copy of the PCDH15/CDH23 heterotetramer and by extension, might include multiple MT complexes.

Anisotropy in mechanical unfolding of protein upon partner-assisted pulling and handle-assisted pulling

Proteins as force-sensors respond to mechanical cues and regulate signaling in physiology. Proteins commonly connect the source and response points of mechanical cues in two conformations, independent proteins in end-to-end geometry and protein complexes in handshake geometry. The force-responsive property of independent proteins in end-to-end geometry is studied extensively using single-molecule force spectroscopy (SMFS). The physiological significance of the complex conformations in force-sensing is often disregarded as mere surge protectors. However, with the potential of force-steering, protein complexes possess a distinct mechano-responsive property over individual force-sensors. To decipher, we choose a force-sensing protein, cadherin-23, from tip-link complex and perform SMFS using end-to-end geometry and handshake complex geometry. We measure higher force-resilience of cadherin-23 with preferential shorter extensions in handshake mode of pulling over the direct mode. The handshake geometry drives the force-response of cadherin-23 through different potential-energy landscapes than direct pulling. Analysis of the dynamic network structure of cadherin-23 under tension indicates narrow force-distributions among residues in cadherin-23 in direct pulling, resulting in low force-dissipation paths and low resilience to force. Overall, the distinct and superior mechanical responses of cadherin-23 in handshake geometry than single protein geometry highlight a probable evolutionary drive of protein-protein complexes as force-conveyors over independent ones.

Cis-clustering of cadherin-23 controls the kinetics of cell-cell adhesion

Cis and trans-interactions in cadherins are the foundations of cellular adhesions in multicellular organisms. While the trans-interactions mediate the intercellular attachment, the cis-interaction is presumed as reinforcement to trans. Thus, trans precedes cis has been the well-accepted model in cadherin adhesion. The stronger affinity of trans-binding over cis has been the decisive influence in the trans first model. Here we show that cadherin-23, a non-classical cadherin with an extended extracellular region, can undergo cis-clustering in solution independent of trans and phase separate as liquid droplets. Using single-molecule measurements, we decipher that weaker cis-interactions favor the cis-clustering. In-cellulo, the cis-clustering is manifested as puncta, a common feature in non-classical cadherin junctions, and accelerates the cell adhesion. The cis-clustering thus kinetically controls cell-adhesion before trans-binding. Notably, M2-macrophages predominantly express cadherin-23 and rapidly attach to circulatory tumor cells during metastatic migration. However, the relation of cis-clustering with rapid cell-cell adhesion in physiology is not yet established

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 β-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 β-strand-rich proteins.

Fast recovery of disrupted tip links induced by mechanical displacement of hair bundles

Hearing and balance rely on the capacity of mechanically sensitive hair bundles to transduce vibrations into electrical signals that are forwarded to the brain. Hair bundles possess tip links that interconnect the mechanosensitive stereocilia and convey force to the transduction channels. A dimer of dimers, each of these links comprises two molecules of protocadherin 15 (PCDH15) joined to two of cadherin 23 (CDH23). The “handshake” that conjoins the four molecules can be disrupted in vivo by intense stimulation and in vitro by exposure to Ca2+chelators. Using hair bundles from the rat’s cochlea and the bullfrog’s sacculus, we observed that extensive recovery of mechanoelectrical transduction, hair bundle stiffness, and spontaneous bundle oscillation can occur within seconds after Ca2+chelation, especially if hair bundles are deflected toward their short edges. Investigating the phenomenon in a two-compartment ionic environment that mimics natural conditions, we combined iontophoretic application of a Ca2+chelator to selectively disrupt the tip links of individual frog hair bundles with displacement clamping to control hair bundle motion and measure forces. Our observations suggest that, after the normal Ca2+concentration has been restored, mechanical stimulation facilitates the reconstitution of functional tip links.