scholarly journals Nanomechanics of tip-link cadherins

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Javier Oroz ◽  
Albert Galera-Prat ◽  
Rubén Hervás ◽  
Alejandro Valbuena ◽  
Débora Fernández-Bravo ◽  
...  
Keyword(s):  
Single Molecule ◽  
Head Movements ◽  
Functional Adaptation ◽  
Mechanical Stimuli ◽  
Sound Waves ◽  
Remarkable Case ◽  
Mechanoelectrical Transduction ◽  
Tip Link ◽  
Protocadherin 15 ◽  
Cadherin 23

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.

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

2021 ◽  
Author(s):  
Johannes Elferich ◽  
Sarah Clark ◽  
Jingpeng Ge ◽  
April Goehring ◽  
Aya Matsui ◽  
...  
Keyword(s):  
Hair Cells ◽  
Electron Tomography ◽  
Electrical Signal ◽  
Mechanical Stimuli ◽  
Microscopy Imaging ◽  
Mechanosensory Transduction ◽  
Composition And Structure ◽  
Tip Link ◽  
Protocadherin 15 ◽  
Cadherin 23

AbstractMechanosensory 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.

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

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Johannes Elferich ◽  
Sarah Clark ◽  
Jingpeng Ge ◽  
April Goehring ◽  
Aya Matsui ◽  
...  
Keyword(s):  
Hair Cells ◽  
Electron Tomography ◽  
Electrical Signal ◽  
Molecular Structures ◽  
Mechanical Stimuli ◽  
Microscopy Imaging ◽  
Mechanosensory Transduction ◽  
Tip Link ◽  
Protocadherin 15 ◽  
Cadherin 23

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.

scholarly journals Structure of mouse protocadherin 15 of the stereocilia tip link in complex with LHFPL5

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Jingpeng Ge ◽  
Johannes Elferich ◽  
April Goehring ◽  
Huaying Zhao ◽  
Peter Schuck ◽  
...  
Keyword(s):  
Ion Channel ◽  
Fold Axis ◽  
Mechanical Stimuli ◽  
Transmembrane Helices ◽  
Membrane Bilayer ◽  
Electrical Signals ◽  
Tip Link ◽  
Protocadherin 15 ◽  
Cadherin 23

Hearing and balance involve the transduction of mechanical stimuli into electrical signals by deflection of bundles of stereocilia linked together by protocadherin 15 (PCDH15) and cadherin 23 ‘tip links’. PCDH15 transduces tip link tension into opening of a mechano-electrical transduction (MET) ion channel. PCDH15 also interacts with LHFPL5, a candidate subunit of the MET channel. Here we illuminate the PCDH15-LHFPL5 structure, showing how the complex is composed of PCDH15 and LHFPL5 subunit pairs related by a 2-fold axis. The extracellular cadherin domains define a mobile tether coupled to a rigid, 2-fold symmetric ‘collar’ proximal to the membrane bilayer. LHFPL5 forms extensive interactions with the PCDH15 transmembrane helices and stabilizes the overall PCDH15-LHFPL5 assembly. Our studies illuminate the architecture of the PCDH15-LHFPL5 complex, localize mutations associated with deafness, and shed new light on how forces in the PCDH15 tether may be transduced into the stereocilia membrane.

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

2020 ◽  
Vol 117 (40) ◽  
pp. 24837-24848
Author(s):  
Deepanshu Choudhary ◽  
Yoshie Narui ◽  
Brandon L. Neel ◽  
Lahiru N. Wimalasena ◽  
Carissa F. Klanseck ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Inner Ear ◽  
Sensory Perception ◽  
Head Movements ◽  
Structural Determinants ◽  
Tip Link ◽  
Protocadherin 15 ◽  
Cadherin 23 ◽  
Dynamics Simulations

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.

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

2020 ◽  
Author(s):  
R. G. Alonso ◽  
M. Tobin ◽  
P. Martin ◽  
A. J. Hudspeth
Keyword(s):  
Electrical Response ◽  
Ionic Channels ◽  
Hair Bundle ◽  
Mechanical Stimuli ◽  
Mechanoelectrical Transduction ◽  
Hair Bundles ◽  
Protocadherin 15 ◽  
Cadherin 23

AbstractHearing 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 towards 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.Significance StatementEach of the sensory receptors responsible for hearing or balance—a hair cell—has a mechanosensitive hair bundle. Mechanical stimuli pull upon molecular filaments—the tip links—that open ionic channels in the hair bundle. Loud sounds can damage hearing by breaking the tip links; recovery by replacement of the constituent proteins then requires several hours. We disrupted the tip links in vitro by removing the calcium ions that stabilize them, then monitored the electrical response or stiffness of hair bundles to determine whether the links could recover. We found that tip links recovered within seconds if their ends were brought back into contact. This form of repair might occur in normal ears to restore sensitivity after damage.

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

2020 ◽  
Vol 117 (48) ◽  
pp. 30722-30727
Author(s):  
R. G. Alonso ◽  
M. Tobin ◽  
P. Martin ◽  
A. J. Hudspeth
Keyword(s):  
Hair Bundle ◽  
Fast Recovery ◽  
Mechanoelectrical Transduction ◽  
Hair Bundles ◽  
Protocadherin 15 ◽  
Cadherin 23 ◽  
The Brain ◽  
Intense Stimulation

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.

scholarly journals A Mechanically Weak Extracellular Membrane-Adjacent Domain Induces Dimerization of Protocadherin-15

2018 ◽  
Author(s):  
P. De-la-Torre ◽  
D. Choudhary ◽  
R. Araya-Secchi ◽  
Y. Narui ◽  
M. Sotomayor
Keyword(s):  
Protein Interactions ◽  
Analytical Ultracentrifugation ◽  
Size Exclusion ◽  
Molecular Architecture ◽  
Protein Protein Interactions ◽  
Tip Link ◽  
Exclusion Chromatography ◽  
Protocadherin 15 ◽  
Cadherin 23 ◽  
Dynamics Simulations

ABSTRACTThe cadherin superfamily of proteins is defined by the presence of extracellular cadherin (EC) repeats that engage in protein-protein interactions to mediate cell-cell adhesion, cell signaling, and mechanotransduction. The extracellular domains of non-classical cadherins often have a large number of EC repeats along with other subdomains of various folds. Protocadherin-15 (PCDH15), a protein component of the inner-ear tip link filament essential for mechanotransduction, has eleven EC repeats and a membrane adjacent domain (MAD12) of atypical fold. Here we report the crystal structure of a pig PCDH15 fragment including EC10, EC11, and MAD12 in a parallel dimeric arrangement. MAD12 has a unique molecular architecture and folds as a ferredoxin-like domain similar to that found in the nucleoporin protein Nup54. Analytical ultracentrifugation experiments along with size exclusion chromatography coupled to multi-angle laser light scattering and small-angle X-ray scattering corroborate the crystallographic dimer and show that MAD12 induces parallel dimerization of PCDH15 near its membrane insertion point. In addition, steered molecular dynamics simulations suggest that MAD12 is mechanically weak and may unfold before tip-link rupture. Sequence analyses and structural modeling predict the existence of similar domains in cadherin-23, protocadherin-24, and the “giant” FAT and CELSR cadherins, indicating that some of them may also exhibit MAD-induced parallel dimerization.

scholarly journals Plasma Membrane Targeting of Protocadherin 15 Is Regulated by the Golgi-Associated Chaperone Protein PIST

2016 ◽  
Vol 2016 ◽  
pp. 1-9 ◽  
Author(s):  
Hongyun Nie ◽  
Yueyue Liu ◽  
Xiaolei Yin ◽  
Huiren Cao ◽  
Yanfei Wang ◽  
...  
Keyword(s):  
Pdz Domain ◽  
Proper Function ◽  
Membrane Targeting ◽  
Membrane Expression ◽  
Binding Interface ◽  
Chaperone Protein ◽  
Tip Link ◽  
Protocadherin 15 ◽  
Cadherin 23 ◽  
Golgi Network

Protocadherin 15 (PCDH15) is a core component of hair cell tip-links and crucial for proper function of inner ear hair cells. Mutations of PCDH15 gene cause syndromic and nonsyndromic hearing loss. At present, the regulatory mechanisms responsible for the intracellular transportation of PCDH15 largely remain unknown. Here we show that PIST, a Golgi-associated, PDZ domain-containing protein, interacts with PCDH15. The interaction is mediated by the PDZ domain of PIST and the C-terminal PDZ domain-binding interface (PBI) of PCDH15. Through this interaction, PIST retains PCDH15 in the trans-Golgi network (TGN) and reduces the membrane expression of PCDH15. We have previously showed that PIST regulates the membrane expression of another tip-link component, cadherin 23 (CDH23). Taken together, our finding suggests that PIST regulates the intracellular trafficking and membrane targeting of the tip-link proteins CDH23 and PCDH15.

scholarly journals Elasticity of individual protocadherin 15 molecules implicates tip links as the gating springs for hearing

2019 ◽  
Vol 116 (22) ◽  
pp. 11048-11056 ◽  
Author(s):  
Tobias F. Bartsch ◽  
Felicitas E. Hengel ◽  
Aaron Oswald ◽  
Gilman Dionne ◽  
Iris V. Chipendo ◽  
...  
Keyword(s):  
Hair Cells ◽  
Essential Feature ◽  
Optical Trap ◽  
Tip Link ◽  
Entropic Spring ◽  
Distinct Frequency ◽  
Low Stiffness ◽  
Stiffness Control ◽  
Protocadherin 15 ◽  
Cadherin 23

Hair cells, the sensory receptors of the inner ear, respond to mechanical forces originating from sounds and accelerations. An essential feature of each hair cell is an array of filamentous tip links, consisting of the proteins protocadherin 15 (PCDH15) and cadherin 23 (CDH23), whose tension is thought to directly gate the cell’s transduction channels. These links are considered far too stiff to represent the gating springs that convert hair bundle displacement into forces capable of opening the channels, and no mechanism has been suggested through which tip-link stiffness could be varied to accommodate hair cells of distinct frequency sensitivity in different receptor organs and animals. Consequently, the gating spring’s identity and mechanism of operation remain central questions in sensory neuroscience. Using a high-precision optical trap, we show that an individual monomer of PCDH15 acts as an entropic spring that is much softer than its enthalpic stiffness alone would suggest. This low stiffness implies that the protein is a significant part of the gating spring that controls a hair cell’s transduction channels. The tip link’s entropic nature then allows for stiffness control through modulation of its tension. We find that a PCDH15 molecule is unstable under tension and exhibits a rich variety of reversible unfolding events that are augmented when the Ca2+ concentration is reduced to physiological levels. Therefore, tip link tension and Ca2+ concentration are likely parameters through which nature tunes a gating spring’s mechanical properties.

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