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

Abstract
Data availability
Article and author information
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Abstract

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.

Data availability

Tomograms depicted in the figures have been deposited in the EMDB under accession 424 codes EMD-25046, EMD-25047, EMD-25048, EMD-25049, EMD-25050, EMD-25051, EMD-425 25052, EMD-25053, EMD-25054, EMD-25055, EMD-25056, EMD-25057, EMD-25058, EMD-426 25059, EMD-25060, and EMD-25061 (in order of appearance). The corresponding tilt-series have been deposited in EMPIAR under accession code EMPIAR-10820. The tilt series of tomograms not depicted in this paper have been deposited in EMPIAR under accession code EMPIAR-10898.

The following data sets were generated

1. Elferich J
2. Clark S
3. Ge J
4. Goehring A
5. Matsui A
6. Gouaux E
(2021) Tomogram of mouse stereocilia containing PCDH15 molecules labeled with 39G7-AuNPs
EMDB: EMD-25047 to EMD-25061.

https://www.ebi.ac.uk/emdb/EMD-25047
1. Elferich
2. J.
3. Clark
4. S.
5. Elferich J
6. Clark S
7. Ge J
8. Goehring A
9. Matsui A
10. Gouaux EGe
11. J.
12. Goehring
13. A.
14. Matsui
15. A.
16. Gouaux
17. E.
(2021) Tomogram of mPCDH15/39G7-AuNP complex
EMDB: EMD-25046.

https://www.ebi.ac.uk/emdb/EMD-25046
1. Elferich
2. J.
3. Clark
4. S.Elferich J
5. Clark S
6. Ge J
7. Goehring A
8. Matsui A
9. Gouaux E
10. Ge
11. J.
12. Goehring
13. A.
14. Matsui
15. A.
16. Gouaux
17. E.
(2021) Molecular structure and conformation of stereocilia tip-links elucidated by cryo-electron tomography
EMPIAR: EMPIAR-10820.

https://www.ebi.ac.uk/empiar/EMPIAR-10820/
1. Elferich
2. J.
3. Clark
4. SElferich J
5. Clark S
6. Ge J
7. Goehring A
8. Matsui A
9. Gouaux E.
10. Ge
11. J.
12. Goehring
13. A.
14. Matsui
15. A.
16. Gouaux
17. E.
(2021) Cryo-electron tilt series of mouse stereocilia
EMPIAR: EMPIAR-10898.

https://www.ebi.ac.uk/empiar/EMPIAR-10898/

Article and author information

Author details

Johannes Elferich

Vollum Institute, Oregon Health and Science University, Portland, United States

Competing interests
The authors declare that no competing interests exist.
Sarah Clark

Vollum Institute, Oregon Health and Science University, Portland, United States

Competing interests
The authors declare that no competing interests exist.
Jingpeng Ge

Vollum Institute, Oregon Health and Science University, Portland, United States

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0001-6164-1221
April Goehring

Vollum Institute, Oregon Health and Science University, Portland, United States

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-2592-6956
Aya Matsui

Vollum Institute, Oregon Health and Science University, Portland, United States

Competing interests
The authors declare that no competing interests exist.
Eric Gouaux

Vollum Institute, Oregon Health and Science University, Portland, United States

For correspondence
gouauxe@ohsu.edu

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-8549-2360

Funding

National Institute of General Medical Sciences (U24GM129547)

Eric Gouaux

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols (TR01_IP00000905) of Oregon Health and Science University. Mice were euthanized according to the American Veterinary Medical Association Guidelines for Euthanasia of Animals before samples were prepared.

Copyright

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.