Molecular structures and conformations of protocadherin-15 and its complexes on stereocilia elucidated by cryo-electron tomography
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Evaluation Summary:
The manuscript by Elferich et al. describes challenging experiments to visualize components of the hair-cell mechanotransduction complex, especially PCDH15, in unfixed, fast-frozen stereocilia. The manuscript presents exquisite images of PCDH15 in its native state, confirming the predictions of earlier studies. While much of what is presented here was predicted from less-direct measurements, it is useful to confirm these with direct imaging of single molecules.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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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.
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Evaluation Summary:
The manuscript by Elferich et al. describes challenging experiments to visualize components of the hair-cell mechanotransduction complex, especially PCDH15, in unfixed, fast-frozen stereocilia. The manuscript presents exquisite images of PCDH15 in its native state, confirming the predictions of earlier studies. While much of what is presented here was predicted from less-direct measurements, it is useful to confirm these with direct imaging of single molecules.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
The submission "Molecule structure and conformation of stereocilia tip-links elucidated by cryo-electron tomography" represents an attempt to better delineate the structure of the molecular apparatus-the transduction complex-by means of which mammalian hair cells respond to mechanical stimuli. In particular, the authors use cryo-electron microscopic tomography to examine unfixed preparations of murine hair bundles that have been pressed against grid films. By labeling protocadherin 15 (PCHD15) with a novel immunogold construct, they demonstrate the presence of dimeric and possibly monomeric PCDH15 molecules interacting with stereociliary membranes, membranous vesicles, and other filamentous structures perhaps including cadherin 23 (CDH23) molecules.
In a broad sense, any filamentous structure that bridges …
Reviewer #1 (Public Review):
The submission "Molecule structure and conformation of stereocilia tip-links elucidated by cryo-electron tomography" represents an attempt to better delineate the structure of the molecular apparatus-the transduction complex-by means of which mammalian hair cells respond to mechanical stimuli. In particular, the authors use cryo-electron microscopic tomography to examine unfixed preparations of murine hair bundles that have been pressed against grid films. By labeling protocadherin 15 (PCHD15) with a novel immunogold construct, they demonstrate the presence of dimeric and possibly monomeric PCDH15 molecules interacting with stereociliary membranes, membranous vesicles, and other filamentous structures perhaps including cadherin 23 (CDH23) molecules.
In a broad sense, any filamentous structure that bridges two stereociliary tips could be termed a "tip link." The conventional definition, though, restricts the term as defined by the authors on lines 21-22. Using the term more broadly risks erroneous conclusions: especially during murine development, there are clearly a wide variety of filamentous structures at stereociliary tips, nearly all of which disappear within the first two weeks of postnatal life. The term "tip link" should be stricken from instances in which the nature of the structures is not rigorously documented. This includes the submission's title and sections such as that beginning at line 196, "Tomographic reconstructions of PCDH15-CDH23 heterotetramers." The authors have not confirmed the identities of such structures, and in particular have not demonstrated the presence of CDH23. The same holds for the section commencing on line 217, "An intact tip-link surrounded by non CDH23-bound PCDH15 molecules." The single structure in question is described as "most likely an intact tip-link," but that degree of proof does not inspire confidence in either the section title or indeed the title of the submission itself.
The assertion on line 253-254 that "this work is the first look at native tip-links in situ" is unsubstantiated: the authors are staking an unjustified claim of priority. Although such an assertion should be supported by several well-documented structures, the actual evidence amounts to one "putative" tip link. Moreover, the structures are scarcely "native," given the violence of the bundle-blotting procedure and the acknowledged damage as a result; nor are they "in situ," which would imply their presence in a mouse's inner ear, or at least in living, functioning hair cells. The authors should describe what they have nicely demonstrated, the immunologically confirmed occurrence of PCDH15 molecules-many of them dimers-near stereociliary tips, and the association of those molecules with other as yet unidentified filamentous and lipidic structures.
The preparative techniques are sound and the tomographic images of generally high quality, as manifested for example by the smooth membrane profiles and nicely ordered actin microfilaments of the stereociliary cytoskeletons. Nevertheless, the intrepretation of the data is shaky. Figure 3C gives striking evidence for a dimer of PCDH15. It is not at all clear, either in the figure or in the associated video, that the structure in panel (B) shows two labels; and that in panel (C) shows a dense blur, perhaps a cluster of gold particles. It is likewise unclear that there is any label bound in panel (D). In view of the ambiguous micrographs, a reader might well be skeptical about the ensuing statistical treatment.
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Reviewer #2 (Public Review):
The manuscript presents exquisite images of PCDH15 in its native state, confirming the predictions of earlier studies. While much of what is presented here was predicted from less-direct measurements, it is gratifying to confirm these with direct imaging of single molecules. Specifically, the paper demonstrates that PCDH15 in its native state at the tip of a stereocilium is a dimer; it shows full tip links with PCDH15 bound to strands likely to be CDH23 with N-terminal binding; it shows that PCDH15 can bend in vivo at a location at or near the EC9-EC10 junction; it shows how that bending could allow a tip link to extend upwards from a location below a stereocilium tip; it shows a protein likely to be CDH23 bending near the middle of its extracellular domain; it confirms that the dense cross links of immature …
Reviewer #2 (Public Review):
The manuscript presents exquisite images of PCDH15 in its native state, confirming the predictions of earlier studies. While much of what is presented here was predicted from less-direct measurements, it is gratifying to confirm these with direct imaging of single molecules. Specifically, the paper demonstrates that PCDH15 in its native state at the tip of a stereocilium is a dimer; it shows full tip links with PCDH15 bound to strands likely to be CDH23 with N-terminal binding; it shows that PCDH15 can bend in vivo at a location at or near the EC9-EC10 junction; it shows how that bending could allow a tip link to extend upwards from a location below a stereocilium tip; it shows a protein likely to be CDH23 bending near the middle of its extracellular domain; it confirms that the dense cross links of immature bundles do contain PCDH15; and it give some sense of the number of PCDH15 molecules on each stereocilium.
Overall, the claims are supported by the images. The proteins of interest are at the limit of resolution of the cryo tomography, so we do have to trust that the "annotated" tomograms-really the authors' conception of the tomograms-are fair representations. At the same time, studying over 500 tomograms probably trained the authors' eyes to the point that they could see detail that a first-time viewer cannot.
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