Hearing is a dynamic and delicate connection between protein filaments

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The sense of hearing is, quite literally, a molecular tightrope act. Turns out, it involves acrobatics as well.

In a paper published in Nature Communications on Feb 8, researchers at Harvard Medical School and Boston Children’s Hospital show that a dynamic and delicate connection between two pairs of diminutive protein filaments plays a central role in in hearing.

The tension held by these filaments, together called a tip link, is essential for the activation of sensory cells in the inner ear. The team’s analyses reveal that the filaments, which are joined end-to-end, work together like trapeze artists holding hands. Their grasp on each other can be disrupted, by a loud noise, for example. But with a two-handed grip, they can quickly reconnect when one hand slips.

The findings present a new understanding of the molecular underpinnings of hearing, as well as the sense of balance, which arises from similar processes in the inner ear.

Disorders of deafness and balance have been linked to mutations in tip links, and the study results could lead to new therapeutic strategies for such disorders, according to the authors.

“This tiny apparatus, made of less than a dozen proteins, is what helps change sound from a mechanical stimulus into an electrical signal that the brain can decipher,” said co-corresponding author David Corey, the Bertarelli Professor of Translational Medical Science at HMS. “Understanding how these proteins work provides insights into the secrets of the sensation of sound.”

The dynamic connection between the filaments may also function as a circuit breaker that protects other cellular components, according to the researchers.

“I think our study gives us a sense of awe for how perfectly engineered this system in the ear is,” said co-corresponding author Wesley Wong, HMS associate professor of biological chemistry and molecular pharmacology at Boston Children’s.

“It maintains a delicate balance between being just strong enough to carry out its function but weak enough to break to potentially preserve the function of other elements that can’t be as easily reformed.”

Decoding the handshake

For hearing to occur, cells must detect and translate pressure waves in the air into bioelectrical signals. This task falls upon hair cells, the sensory cells of the inner ear. Protruding from these cells are bundles of hair-like structures, which bend back and forth as pressure waves move through the inner ear.

Tip link filaments physically connect each hair to another and are anchored onto specialized ion channels. As the bundle moves, the tension of the tip links changes, opening and closing the channels like a gate to allow electric current to enter the cell. In this way, tip links initiate the bioelectrical signals that the brain ultimately processes as sound.

In previous studies, Corey and colleagues explored the composition of tip links and identified the precise atomic structure of the bond between the two protein filaments. Intriguingly, this bond was evocative of a molecular handshake, according to the authors.

In the current study, Corey, Wong and the team set out to understand the nature of this handshake. To do so, they applied single-molecule force spectroscopy, a technique that often uses optical tweezers—highly focused laser beams that can hold extremely small objects and move them by distances as short as a billionth of a meter.

The researchers, led by study first authors Eric Mulhall and Andrew Ward, both research fellows in neurobiology in the Blavatnik Institute at HMS, coated microscopic glass beads with strands of either protocadherin-15 or cadherin-23, the two proteins that make up the tip link. Using optical tweezers, they moved beads close to each other until the protein strands stuck together end to end and then measured the forces needed to pull the bonds apart.

Stronger than the sum

Each tip link is made up of two strands of both proteins. The team found that the strength of this double-stranded bond far surpassed the strength of the bond between individual strands of either protein. Under low tension, a double-stranded bond lasted ten times longer than a single-stranded bond before breaking.

This increased strength appears to be due to the dynamic nature of the connection, according to the authors. Rather than acting as a simple static rope, the filaments detach and reattach to each other within tenths of a second. A force may break one pair of strands apart, but the other pair can remain connected long enough for the broken pair to rejoin.

At extremely high forces, however, the double-stranded bond breaks rapidly. This feature may help to prevent catastrophic damage to other components of the hair cell, the authors said.

“If the tip link were super strong, then when exposed to a very loud sound it might rip the whole complex out of the cell membrane, which would be hard to recover from,” said Wong, who is also an associate faculty member at the Wyss Institute for Biologically Inspired Engineering at Harvard.

“The ability to break with loud sounds is analogous to a mechanical circuit breaker,” he added. “This use of multiple weak bonds to form a tunable biological circuit breaker could potentially be very interesting for synthetically engineered systems.”

Surprisingly, the team found that under resting tension, each tip link lasts only around eight seconds before it breaks. Their analyses, coupled with evidence from other studies, suggest that new tip links can form rapidly from other strands of protein nearby. Together, the results support a new paradigm of highly dynamic tip link formation and rupture that both enables and protects hearing.

The team also looked at mutations to protocadherin-15 that are linked to Usher syndrome, a rare hereditary disorder of deafness and blindness. Their experiments suggest that some of these mutations can greatly weaken the bond between the tip link filaments. This may be why the disorder leads to deafness, and further mechanistic understanding of this process could lead to new therapeutic approaches, the authors said.

“It’s hard to fix something if you don’t really know what’s broken, and we are optimistic that a better understanding can help lead to new solutions,” Corey said.

In addition, the new findings may help inform study in other areas of the body.

“We have many different mechanical senses besides hearing, such as touch, the sensation of blood pressure, and certain types of pain,” Corey added. “We understand hearing in more molecular detail than any of the others – knowledge that can help us probe the workings of other mechanical senses.”


Inner-ear sensory perception begins when hair-cell mechanosensitive ion channels (1–3) are gated by “tip links,” fine protein filaments essential for hearing and balance (Fig. 1A) (4–7). Tip links are 150 to 180 nm long (8–10), their integrity is calcium (Ca2+)-dependent (5, 11), and they are formed by protocadherin-15 (PCDH15; ∼200 kDa) and cadherin-23 (CDH23; ∼370 kDa) (12, 13), two large proteins involved in hereditary deafness (14, 15), audiogenic seizures (16), and progressive blindness (17). Mature tip links are thought to be heterotetramers formed by parallel (cis) homodimers of PCDH15 interacting in an antiparallel trans mode (tip-to-tip) with cis homodimers of CDH23 (13), whereas immature tip links are likely formed by trans interactions of two PCDH15 molecules (18, 19).

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Fig. 1.
Inner-ear mechanotransduction and PCDH15 structures. (A) Hair-cell bundle row showing location of tip links. Force from sound or head movements displaces the bundle to activate the transduction apparatus. (B) The tip link is made of CDH23 (blue) and PCDH15 (purple). (C) Schematics of PCDH15 (Left) and structures of PCDH15 fragments (Right) used to build models of the entire PCDH15 ectodomain. PDB codes are indicated for all 11 structures presented here, with codes in parentheses for three additional structures presented elsewhere (28, 31, 32). All structures are in surface representation, with PCDH15 fragments in purple, CDH23 EC1-2 in blue, Ca2+ ions in green, and glycosylation sugars in red and yellow. Gray arrows in the schematics indicate EC linkers with atypical canonical-like linker regions (EC8-9 in light gray), with partial Ca2+-free linker regions (EC2-3, EC3-4, and EC5-6 in gray), and with Ca2+-free linker regions (EC9-10 and EC11-MAD12 in dark gray).

Some details of the PCDH15 and CDH23 interaction are well understood: Immunogold electron microscopy (EM) suggests that tip-link lengths are mostly consistent with the predicted length of the combined proteins interacting tip-to-tip (8, 13); competitive binding of exogenous PCDH15 and CDH23 tip fragments to endogenous proteins blocks regeneration of tip links and associated transduction currents during hair-cell development and after tip-link rupture with a Ca2+ chelator (20); and structures of the complex formed by the tips of PCDH15 and CDH23 engaged in a heterodimeric molecular “handshake” have been obtained and validated in vitro and in vivo (21, 22).

Furthermore, mutations that cause deafness in humans and mice have been shown to or are predicted to break the handshake interaction (21–24). In addition, studies of the CDH23 and PCDH15 heterodimer show that its strength can be tuned by PCDH15 isoforms that have distinct N-terminal tips (25). The strength of the heterotetrameric bond can be further diversified when considering combinations of PCDH15 isoforms in parallel.

However, little was known about the structure of parallel cis dimers formed by PCDH15 until recently (26–28). While negative-staining transmission EM showed conformationally heterogeneous parallel dimers for tip-link components (13), most extracellular fragments of PCDH15 (and CDH23) studied had been monomeric in solution (26, 29–33). Recent crystal structures of PCDH15 fragments (26–28) and a low-resolution EM-based model suggest two points of dimerization (26, 27), yet a detailed atomistic model of the complete ectodomain is still missing. In addition, how PCDH15 engages in trans homodimers is unclear, and the structural details and strength of the PCDH15 + CDH23 heterotetrameric bond are not known.

The oligomerization states of PCDH15 and CDH23 and the architecture of their heterotetrameric bond are important determinants of tip-link mechanics and inner-ear mechanotransduction (10, 26). Disruption of either the complex between PCDH15 and CDH23 or the oligomerization of PCDH15 impairs transduction (21, 22, 26). The inner-ear transduction channel is gated by a soft element called the “gating spring,” which is either in series with the tip link or the tip link itself (2, 3, 34–36). Whether cadherin tip links are elastic or rigid remains controversial (6, 10, 30, 31).

The ultrastructure of tip links suggested a stiff elastic element (6), but the length of tip links varies in situ (8). PCDH15 and CDH23 feature 11 and 27 extracellular cadherin (EC) “repeats” (Fig. 1B), respectively, and initial studies of the structure and simulated dynamics of the CDH23 EC1-2 tip predicted that these canonical repeats and their linker region fully occupied by Ca2+ ions at sites 1, 2, and 3 would be stiff (30). However, while the EC repeats along PCDH15 and CDH23 are predicted to share a common fold, they vary in sequence, which can result in structural heterogeneity, as seen for other long cadherins (37, 38).

For example, crystal structures revealed a bent and L-shaped Ca2+-free PCDH15 EC9-10 linker region, with simulations evincing that unbending can provide some elasticity to the tip link (31). Bending and flexibility at this L-shaped EC9-10 linker region are also observed in low-resolution cryo-EM conformations of a cis dimeric PCDH15 fragment encompassing EC8 down to its transmembrane helix (27). These results highlight the diverse mechanical responses of various tip-link cadherin fragments.

Crystal structures and simulations have also shown that other parts of PCDH15 can be structurally distinct. The PCDH15 EC3-4 linker is flexible and binds two Ca2+ ions, instead of three. In addition, this atypical linker binds Ca2+ ions with decreased binding affinity (45 μM for site 3 and >100 μM for site 2) as compared to the canonical Ca2+-binding linker of CDH23 EC1-2 (5 μM for site 3, 44 μM for site 2, and 71 μM for site 1) (30, 32, 39).

Occupancy of Ca2+-binding sites at EC linkers will greatly determine the mechanics of tip links and whether unfolding of EC repeats could occur before unbinding of the handshake bond (21). Interestingly, bulk endolymphatic Ca2+ concentration in the cochlea is tightly regulated and varies along its length from ∼20 μM (base) to ∼40 μM (apex) (40, 41). However, Ca2+ concentration near cochlear tip links could be significantly larger (42, 43), and vestibular Ca2+ concentrations range from 90 to 150 μM (41, 44). Yet, the structure and the Ca2+-dependent mechanics of entire heterotetrameric tip links remain undetermined.

To better understand the mechanics of tip links and the first steps of inner-ear mechanotransduction, we have determined 11 X-ray crystal structures of PCDH15 fragments (Fig. 1C and SI Appendix, Table S1), including the structure of a PCDH15 EC1-3 + CDH23 EC1-2 heterotetrameric complex depicting a parallel homodimer of PCDH15 interacting with two molecules of CDH23, and 10 other fragments covering all of PCDH15’s EC repeats, all of its EC linkers, and its membrane-adjacent domain MAD12 (28), also referred to as SEA (45), PICA (26), or EL (27).

These structures allowed us to assemble a complete model of the monomeric ectodomain of PCDH15, to suggest models of trans and cis PCDH15 homodimers, and to build a model of the PCDH15–CDH23 bond. In addition, we used steered molecular dynamics (SMD) (46) simulations to predict the Ca2+-dependent mechanics of these models, which suggest a multimodal (stiff or soft) elastic response for PCDH15 and indicates some conditions in which PCDH15 can provide both the elasticity and extensibility typically associated with the hair-cell gating spring.

DISCUSSION
Our structures, biochemical data, and simulations provide an integrated atomistic view of the entire PCDH15 ectodomain and its possible modes of homophilic and heterophilic interaction (Fig. 7A). We found two points of parallel dimerization for PCDH15 (at EC2-3 and EC11-MAD12) and provide a detailed and unique view of the two trans PCDH15-CDH23 handshakes in the heterotetrameric bond facilitated by the PCDH15 EC2-3 X-dimer.

Simulations suggest that the two handshakes in the heterotetramer are squeezed together by closing of the X-dimer scissor when tension that mimics physiological stimuli is applied. This squeezing strengthens the heterotetrameric bond with unbinding forces predicted to be on average ∼1.3 times larger than the sum of unbinding forces for independent handshakes, reminiscent of catch-bond behavior where the dissociation lifetime of a bond is increased by tensile forces (63). The dual handshake may also facilitate, through avidity, a strengthened mechanical response and extended lifetime of the tip-link bond (64).

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Fig. 7.
Interaction modes and elastic behavior of PCDH15. (A) Potential homophilic and heterophilic interactions of PCDH15 (mauve and purple) and CDH23 (cyan and blue) suggested by structural analyses. Top arrangement could be adopted by kinociliary links (56), while the middle heterophilic complex is compatible with the heterotetrameric tip link (13, 21). The two remaining homophilic arrangements shown at the bottom are possible homophilic trans interactions in immature tip links (18, 19). (B) Structures and simulations indicate that the PCDH15 ectodomain can have distinct elastic responses that are tuned by tension and Ca2+. Phase diagram shows possible hypothetical states for PCDH15’s ectodomain. At very low Ca2+ concentrations (Upper Left) PCDH15 will not interact with CDH23 and its linkers will be flexible and easily stretchable under tension. At high tension and very low Ca2+ concentrations (Lower Left), the Ca2+-free linkers will be weak and unfolding (uf) at any of the EC repeats will ensue. Under normal physiological conditions (blue shade), PCDH15 will form a cis dimer that interacts with CDH23 and that can exhibit soft elasticity mediated by bending and unbending transitions of Ca2+-free linker regions EC5-6 and EC9-10. As tension increases, unbending (ube) results in stiffening of PCDH15. Stretching of linkers (s) and unrolling of MAD12 (ur) at higher tension may soften the elastic response of PCDH15 for the subsequent stimulus. Under more extreme tension (Lower Right) unbinding of PCDH15 from CDH23 (ubi) or unfolding of PCDH15 (uf) may occur. Glycosylation and specifics of PCDH15 splice isoforms may also influence the tip-link state and elastic response. The effect of resting tension (58, 59) is further discussed in SI Appendix, Fig. S16.

Our structures also unmasked three points of Ca2+-independent flexibility for PCDH15 at linker regions EC3-4, EC5-6, and EC9-10. Simulations of the entire monomeric PCDH15 ectodomain bound to CDH23 EC1-2/3 revealed a soft elastic response dominated by these flexible points, while the heterotetrameric complex (dimeric PCDH15 bound to two CDH23 monomers) was stiffer yet still presented kinks at the PCDH15 EC9-10 linker region that could provide some limited extensibility. Intriguingly, our simulations predict that PCDH15’s MAD12 unrolls and then unfolds before CDH23 unbinding at the stretching speeds (down to 0.02 nm/ns).

Unfolding of MADs in PCDH15 and CDH23 (28) could explain uncompromised hair-cell mechanotransduction under extreme stimuli that would require large tip-link extensibility (∼100 nm) (65, 66). These results highlight the potential complexities of the tip-link mechanical response and provide a structural framework to both compare and interpret complementary experimental results (26, 27, 59, 64, 67–69) and to understand the function of PCDH15 as a key component of the tip links that open inner-ear transduction channels (SI Appendix, Note 4).

In vivo conditions for tip links vary significantly across organs and species. For example, Ca2+ concentration near tip links will greatly depend on the organ in which hair cells are located. The vestibular endolymph Ca2+ concentration is 90 to 150 μM, while the bulk cochlear endolymph Ca2+ concentration varies from 20 to 40 μM (40, 41).

Intriguingly, Ca2+ concentrations near hair bundles in the cochlear subtectorial space might be significantly higher than previously thought (>300 μM), both because of the action of stereocilia Ca2+ pumps functioning in a restricted space and because of the buffering effect of the tectorial membrane (42, 43). Similarly, resting tension and physiological forces will vary within and across organs, where glycosylation and differential expression of various tip-link PCDH15 isoforms may also be diverse.

The complexity of PCDH15’s ectodomain revealed by our structures and simulations and the various sets of environments in which it functions in mechanotransduction indicate that PCDH15 might be a versatile and multimodal protein that can be tuned to display distinct elastic responses (Fig. 7B and SI Appendix, Fig. S16). While under some conditions PCDH15 may display the soft elasticity and extensibility attributed to gating springs, including significant extensibility through MAD12 unfolding, directly determining the exact in vivo conditions in which PCDH15 functions remains a necessary and challenging step required to fully comprehend the role played by tip links in inner-ear mechanotransduction.

PCDH15 is also found in auditory cortex interneurons (16), eye photoreceptors (52, 70), and various other tissues (15), where it may play a role in cell–cell adhesion and tissue development and maintenance. PCDH15’s interaction with CDH23 seems to be essential for auditory cortex wiring (16), while its specific role in photoreceptor function is less clear. Our PCDH15 structures and biochemical assays probing mutations that impair heterophilic binding to CDH23, cis dimerization, and Ca2+ binding provide data that can inform the exploration of PCDH15’s function beyond inner-ear mechanotransduction.

reference link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7547225/


More information: Nature Communications (2021). DOI: 10.1038/s41467-021-21033-

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