Single-molecule force spectroscopy reveals the dynamic strength of the hair-cell tip-link connection

Mulhall, Eric M.; Ward, Andrew; Yang, Darren; Koussa, Mounir A.; Corey, David P.; Wong, Wesley P.

doi:10.1038/s41467-021-21033-6

Cited by 26 publications

(31 citation statements)

References 102 publications

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“…It is also possible that a large number of PCDH15 molecules may be necessary for rebinding to CDH23 in the event of tip link breakage. Atomic force microscopy experiments indicate that the lifetime of the PCDH15-CDH23 bond is only ~8 seconds at resting tension, suggesting that the tip link is a highly dynamic connection (Mulhall et al, 2021). A pool of nearby PCDH15 molecules may enable fast recovery after tip links are broken.…”

Section: Discussionmentioning

confidence: 99%

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

Elferich

Clark

et al. 2021

eLife

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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.

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Section: Discussionmentioning

confidence: 99%

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

Elferich

Clark

et al. 2021

eLife

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“…The specificity of this interaction is such that a mutation in a single residue at the interface can abolish it completely, leading to a high level of conservation at such sites. Together, these results and biophysical experiments (Mulhall et al 2021) revealed that the CDH23-PCDH15 interaction is strong enough to withstand the physiological forces that it would be subjected to in the inner ear. Furthermore, CDH23 and PCDH15 have been identified in zebrafish and chicken hair cells (Söllner et al 2004;Seiler et al 2005;Goodyear et al 2010), suggesting that this interaction and its role in inner-ear mechanotransduction is conserved among vertebrates.…”

Section: Introductionmentioning

confidence: 69%

“…While the CDH23-PCDH15 EC1-2 dimer is ultimately responsible for force transduction, it does so in the context of other structural features that may exhibit functionally significant species-specific changes. The PCDH15 X-dimer, formed at an EC2-3 interface, and an interaction facilitated by MAD12, are two points of dimerization in PCDH15 that have functional implications upon the application of force (De-la-Torre et al 2018;Dionne et al 2018;Ge et al 2018;Choudhary et al 2020;Mulhall et al 2021), and may have evolved to respond to different frequencies.…”

Section: Discussionmentioning

confidence: 99%

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

Nisler

Narui

Choudhary

et al. 2022

Preprint

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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 are 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 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, while subsequent neutral evolution combined with varying degrees of purifying selection drove additional diversification in modern tetrapods.

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“…For each histogram, we construct frequency distribution using the Kernel density smoothing function (in MATLAB). For the Kernel density estimation, we determined the bandwidth using the following equation 46 , 74 – 76 H: bandwidth, n: number of measurements and σ = min{σ x , IQR/1.34} where σ x : standard deviation and IQR is the interquartile range.…”

Section: Methodsmentioning

confidence: 99%

“…These two proteins as tip-links receive tensile forces of varying magnitudes ranging from 10 to 100 pN from sound-stimuli and convey the force to ion channels during mechanotransduction in hearing. Interestingly, the lifetime of the tip-links complex is measured at varying tensile forces using PAP-based SMFS 46 ; however, the force-responsive behaviour of the constituent cadherins has conveniently been measured using HAP configuration 47 , 48 . Whether the complex configuration of tip-link has any implication on the force-response of constituent cadherins is thus unclear.…”

Section: Introductionmentioning

confidence: 99%

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

2021

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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.

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Single-molecule force spectroscopy reveals the dynamic strength of the hair-cell tip-link connection

Cited by 26 publications

References 102 publications

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

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

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

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

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