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.

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

10.1101/2021.10.01.462800 ◽

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.

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The Mechanosensory Transduction Machinery in Inner Ear Hair Cells

Annual Review of Biophysics ◽

10.1146/annurev-biophys-062420-081842 ◽

2020 ◽

Vol 50 (1) ◽

Author(s):

Wang Zheng ◽

Jeffrey R. Holt

Keyword(s):

Hair Cell ◽

Inner Ear ◽

Hair Cells ◽

Annual Review ◽

Publication Date ◽

Fusion Partner ◽

Mechanical Stimuli ◽

Physical Stimulation ◽

Mechanosensory Transduction ◽

Protocadherin 15

Sound-induced mechanical stimuli are detected by elaborate mechanosensory transduction (MT) machinery in highly specialized hair cells of the inner ear. Genetic studies of inherited deafness in the past decades have uncovered several molecular constituents of the MT complex, and intense debate has surrounded the molecular identity of the pore-forming subunits. How the MT components function in concert in response to physical stimulation is not fully understood. In this review, we summarize and discuss multiple lines of evidence supporting the hypothesis that transmembrane channel-like 1 is a long-sought MT channel subunit. We also review specific roles of other components of the MT complex, including protocadherin 15, cadherin 23, lipoma HMGIC fusion partner-like 5, transmembrane inner ear, calcium and integrin-binding family member 2, and ankyrins. Based on these recent advances, we propose a unifying theory of hair cell MT that may reconcile most of the functional discoveries obtained to date. Finally, we discuss key questions that need to be addressed for a comprehensive understanding of hair cell MT at molecular and atomic levels. Expected final online publication date for the Annual Review of Biophysics, Volume 50 is May 6, 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

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Faculty Opinions recommendation of Cadherin 23 and protocadherin 15 interact to form tip-link filaments in sensory hair cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1092088.544638 ◽

2007 ◽

Author(s):

Peter Gillespie

Keyword(s):

Hair Cells ◽

Sensory Hair ◽

Sensory Hair Cells ◽

Tip Link ◽

Protocadherin 15 ◽

Cadherin 23

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

eLife ◽

10.7554/elife.38770 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 26

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.

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Nanomechanics of tip-link cadherins

Scientific Reports ◽

10.1038/s41598-019-49518-x ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 4

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.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1902163116 ◽

2019 ◽

Vol 116 (22) ◽

pp. 11048-11056 ◽

Cited By ~ 20

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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Cadherin 23 and protocadherin 15 interact to form tip-link filaments in sensory hair cells

Nature ◽

10.1038/nature06091 ◽

2007 ◽

Vol 449 (7158) ◽

pp. 87-91 ◽

Cited By ~ 432

Author(s):

Piotr Kazmierczak ◽

Hirofumi Sakaguchi ◽

Joshua Tokita ◽

Elizabeth M. Wilson-Kubalek ◽

Ronald A. Milligan ◽

...

Keyword(s):

Hair Cells ◽

Sensory Hair ◽

Sensory Hair Cells ◽

Tip Link ◽

Protocadherin 15 ◽

Cadherin 23

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Faculty Opinions recommendation of Cadherin 23 and protocadherin 15 interact to form tip-link filaments in sensory hair cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1092088.545455 ◽

2007 ◽

Author(s):

Guy Richardson

Keyword(s):

Hair Cells ◽

Sensory Hair ◽

Sensory Hair Cells ◽

Tip Link ◽

Protocadherin 15 ◽

Cadherin 23

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The elasticity of individual protocadherin 15 molecules implicates cadherins as the gating springs for hearing

10.1101/503029 ◽

2018 ◽

Cited By ~ 2

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. As a consequence, 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 monomersof 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. Tip-link tension and Ca2+ concentration are therefore likely parameters through which nature tunes a gating spring's mechanical properties.

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