Molecular structures and conformations of protocadherin-15 and its complexes on stereocilia elucidated by cryo-electron tomography
- Oregon Health and Science University, United States
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.
-
Tomogram of mouse stereocilia containing PCDH15 molecules labeled with 39G7-AuNPsEMDB: EMD-25047 to EMD-25061.
-
Cryo-electron tilt series of mouse stereociliaEMPIAR: EMPIAR-10898.
Article and author information
Author details
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.
Reviewing Editor
- Edward H Egelman, University of Virginia, United States
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.
Version history
- Preprint posted: October 1, 2021 (view preprint)
- Received: October 7, 2021
- Accepted: December 27, 2021
- Accepted Manuscript published: December 29, 2021 (version 1)
- Version of Record published: January 19, 2022 (version 2)
Copyright
© 2021, Elferich et al.
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.
Metrics
-
- 2,983
- views
-
- 491
- downloads
-
- 8
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Molecular structures and conformations of protocadherin-15 and its complexes on stereocilia elucidated by cryo-electron tomography
eLife 10:e74512.
https://doi.org/10.7554/eLife.74512
Further reading
-
- Neuroscience
Peripheral neurons are heterogeneous and functionally diverse, but all share the capability to switch to a pro-regenerative state after nerve injury. Despite the assumption that the injury response is similar among neuronal subtypes, functional recovery may differ. Understanding the distinct intrinsic regenerative properties between neurons may help to improve the quality of regeneration, prioritizing the growth of axon subpopulations to their targets. Here, we present a comparative analysis of regeneration across four key peripheral neuron populations: motoneurons, proprioceptors, cutaneous mechanoreceptors, and nociceptors. Using Cre/Ai9 mice that allow fluorescent labeling of neuronal subtypes, we found that nociceptors showed the greater regeneration after a sciatic crush, followed by motoneurons, mechanoreceptors, and, finally, proprioceptors. By breeding these Cre mice with Ribotag mice, we isolated specific translatomes and defined the regenerative response of these neuronal subtypes after axotomy. Only 20% of the regulated genes were common, revealing a diverse response to injury among neurons, which was also supported by the differential influence of neurotrophins among neuron subtypes. Among differentially regulated genes, we proposed MED12 as a specific regulator of the regeneration of proprioceptors. Altogether, we demonstrate that the intrinsic regenerative capacity differs between peripheral neuron subtypes, opening the door to selectively modulate these responses.
-
- Cell Biology
- Neuroscience
Mutations in Drosophila Swiss cheese (SWS) gene or its vertebrate orthologue neuropathy target esterase (NTE) lead to progressive neuronal degeneration in flies and humans. Despite its enzymatic function as a phospholipase is well established, the molecular mechanism responsible for maintaining nervous system integrity remains unclear. In this study, we found that NTE/SWS is present in surface glia that forms the blood-brain barrier (BBB) and that NTE/SWS is important to maintain its structure and permeability. Importantly, BBB glia-specific expression of Drosophila NTE/SWS or human NTE in the sws mutant background fully rescues surface glial organization and partially restores BBB integrity, suggesting a conserved function of NTE/SWS. Interestingly, sws mutant glia showed abnormal organization of plasma membrane domains and tight junction rafts accompanied by the accumulation of lipid droplets, lysosomes, and multilamellar bodies. Since the observed cellular phenotypes closely resemble the characteristics described in a group of metabolic disorders known as lysosomal storage diseases (LSDs), our data established a novel connection between NTE/SWS and these conditions. We found that mutants with defective BBB exhibit elevated levels of fatty acids, which are precursors of eicosanoids and are involved in the inflammatory response. Also, as a consequence of a permeable BBB, several innate immunity factors are upregulated in an age-dependent manner, while BBB glia-specific expression of NTE/SWS normalizes inflammatory response. Treatment with anti-inflammatory agents prevents the abnormal architecture of the BBB, suggesting that inflammation contributes to the maintenance of a healthy brain barrier. Considering the link between a malfunctioning BBB and various neurodegenerative diseases, gaining a deeper understanding of the molecular mechanisms causing inflammation due to a defective BBB could help to promote the use of anti-inflammatory therapies for age-related neurodegeneration.
