Complexes of vertebrate TMC1/2 and CIB2/3 proteins form hair-cell mechanotransduction cation channels

Reviewer #1 (Public Review):

This reviewed preprint is a bit of Frankenstein monster, as it crams together three quite different sets of data. It is essentially three papers combined into one-one paper focused on the role of CIB2/CIB3 in VHCs, one on the role of CIB2/CIB3 in zebrafish, and one on structural modeling of a CIB2/3 and TMC1/2 complex. The authors try to combine the three parts with the overarching theme of demonstrating that CIB2/3 play a functionally conserved role across species and hair cell types, but given the previous work on these proteins, especially Liang et al. (2021) and Wang et al. (2023), this argument doesn't work very well. My sense is that the way the manuscript is written now, the sum is less than the individual parts, and the authors should consider whether the work is better split into three separate papers.

The most important shortcoming is the novelty of the work presented here. In line 89 of the introduction the authors state "However, whether CIB2/3 can function and interact with TMC1/2 proteins across sensory organs, hair-cell types, and species is still unclear." They make a similar statement in the first sentence of the discussion and generally use this claim throughout the paper as motivation for why they performed the experiments. Given the data presented in the Liang et al. (2021) and Wang et al. (2023 papers), however, this statement is not well supported. Those papers clearly demonstrate a role for CIB2/CIB3 in auditory and vestibular cells in mice. Moreover, there is also data in Riazuddin et al. (2012) paper that demonstrates the importance of CIB2 in zebrafish and Drosophila. I think the authors are really stretching to describe the data in the manuscript as novel. Conceptually, it reads more as solidifying knowledge that was already sketched out in the field in past studies.

There is one exception, however, and that is the last part of the manuscript. Here structural studies (AlphaFold 2 modeling, NMR structure determination, and molecular dynamics simulations) bring us closer to the structure of the mammalian TMCs, alone and in complex with the CIB proteins. Moreover, the structural work supports the assignment of the TMC pore to alpha helices 4-7.

Reviewer #2 (Public Review):

The paper 'Complexes of vertebrate TMC1/2 and CIB2/3 proteins 1 form hair-cell mechanotransduction cation channels' by Giese and coworkers is quite an intense reading. The manuscript is packed with data pertaining to very different aspects of MET apparatus function, scales, and events. I have to praise the team that combined molecular genetics, biochemistry, NMR, microscopy, functional physiology, in-vivo tests for vestibulo-ocular reflexes, and other tests for vestibular dysfunction with molecular modeling and simulations. The authors nicely show the way CIBs are associated with TMCs to form functional MET channels. The authors clarify the specificity of associations and elucidate the functional effects of the absence of specific CIBs and their partial redundancy.

Reviewer #3 (Public Review):

This study demonstrates that from fish to mammals CIB2/3 is required for hearing, revealing the high degree of conservation of CIB2/3 function in vertebrate sensory hair cells. The modeling data reveal how CIB2/3 may affect the conductance of the TMC1/2 channels that mediate mechanotransduction, which is the process of converting mechanical energy into an electrical signal in sensory receptors. This work will likely impact future studies of how mechanotransduction varies in different hair cell types.

One caveat is that the experiments with the mouse mutants are confirmatory in nature with regard to a previous study by Wang et al., and the authors use lower resolution tools in terms of function and morphological changes. Another is that the modeling data is not supported by electrophysiological experiments, however, as mentioned above, future experiments may address this weakness.

Author Response

We thank reviewers for their evaluation of our work and their thorough critiques, which we will address in an upcoming revised version of the manuscript. We note that work on mouse and fish CIB knockouts in our laboratories started over a decade ago and our discoveries are contemporary to those recently presented by Liang et al., 2021 and Wang et al., 2023, which we acknowledge, cite, and give credit as appropriate. We also note that work on fish knockouts and on fish Cib3 is completely novel.