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

Giese, Arnaud P. J.; Weng, Wei-Hsiang; Kindt, Katie S.; Chang, Vanessa; Montgomery, Jonathan S.; Ratzan, Evan M.; Beirl, Alisha; Rivera, Roberto Aponte; Lotthammer, Jeffrey M.; Walujkar, Sanket; Foster, Mark P.; Zobeiri, Omid A.; Holt, Jeffrey R.; Riazuddin, Saima; Cullen, Kathleen E.; Sotomayor, Marcos; Ahmed, Zubair M.

doi:10.1101/2023.05.26.542533

Cited by 4 publications

(10 citation statements)

References 104 publications

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“…While there exist a number of non-invasive methods to detect the existence of vestibular dysfunction in mice, such as the rota-rod and balance beam tests 6 , the variety of vestibular-loss circling mouse models suggests screening for circling may serve as a convenient screening tool for identifying novel models of vestibular dysfunction. In the present study, we specifically assessed the circling behavior (Cib2 -/-;Cib3 -/-) dual knockout mice [31][32][33] . CIB2 is found in the stereocilia tips of the receptor cells within the vestibular sensory organs 35 (i.e., vestibular hair cells), suggesting the circling behavior observed in these mice results specifically from deficits in peripheral mechanotransduction.…”

Section: Broader Applicability Of This Approachmentioning

confidence: 99%

“…Five adult wild-type (C57BL/6) mice (Jackson Laboratories) and five adult mutant (Cib2-/-;Cib3-/-) mice were used in this study. Generation of mutant mice is described in a separate paper 33 . Animals were group-housed with their littermates on a 12:12 h light: dark cycle at 20°C with ad libitum access to food and water.…”

Section: Animal Care and Housingmentioning

confidence: 99%

“…Our technique achieves statistical equivalence to individual observers' independent labels at matching ultimate consensus times. Specifically, we assessed (Cib2 -/-;Cib3 -/-) dual knockout mice, a mouse mutant strain we and others have previously reported exhibiting circling [31][32][33]32 . We track the snout and base of the tail in mice using the open-source software package DeepLabCut 4 ("DLC"), then compare the performance of several behavioral detection algorithms which apply straightforward analyses to characteristics of the animals' paths.…”

Section: Introductionmentioning

confidence: 99%

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An Open-Source Tool for Automated Human-Level Circling Behavior Detection

Stanley,

Swaminathan,

Wojahn

et al. 2023

Preprint

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Quantifying behavior and relating it to underlying biological states is of paramount importance in many life science fields. Although barriers to recording postural data have been reduced by progress in deep-learning-based computer vision tools for keypoint tracking, extracting specific behaviors from this data remains challenging. Manual behavior coding, the present gold standard, is labor-intensive and subject to intra- and inter-observer variability. Automatic methods are stymied by the difficulty of explicitly defining complex behaviors, even ones which appear obvious to the human eye. Here, we demonstrate an effective technique for detecting one such behavior, a form of locomotion characterized by stereotyped spinning, termed 'circling'. Though circling has an extensive history as a behavioral marker, at present there exists no standard automated detection method. Accordingly, we developed a technique to identify instances of the behavior by applying simple postprocessing to markerless keypoint data from videos of freely-exploring (Cib2-/-;Cib3-/-) mutant mice, a strain we previously found to exhibit circling. Our technique agrees with human consensus at the same level as do individual observers, and it achieves >90% accuracy in discriminating videos of wild type mice from videos of mutants. As using this technique requires no experience writing or modifying code, it also provides a convenient, noninvasive, quantitative tool for analyzing circling mouse models. Additionally, as our approach was agnostic to the underlying behavior, these results support the feasibility of algorithmically detecting specific, research-relevant behaviors using readily-interpretable parameters tuned on the basis of human consensus.

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Section: Broader Applicability Of This Approachmentioning

confidence: 99%

Section: Animal Care and Housingmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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An Open-Source Tool for Automated Human-Level Circling Behavior Detection

Stanley,

Swaminathan,

Wojahn

et al. 2023

Preprint

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“…We leveraged this data set to further explore the expression of other mechanotransduction components within tmc subtypes. We found specific expression patterns of cib2 and cib3 , which encode calcium binding proteins that interact with TMC1/2 subunits and modulate channel conductance ( Liang et al, 2021 ; Giese et al, 2023 ; Wang et al, 2023 ). Our data indicate that specialization of the mechanotransduction apparatus occurs at the molecular level.…”

Section: Introductionmentioning

confidence: 99%

Differential expression of mechanotransduction complex genes in auditory/vestibular hair cells in zebrafish

Smith,

Sun,

et al. 2023

Front. Mol. Neurosci.

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Ciliated sensory cells such as photo- and olfactory receptors employ multiple types of opsins or hundreds of unique olfactory G-protein coupled receptors to respond to various wavelengths of light or odorants. With respect to hearing and balance, the mechanotransduction machinery involves fewer variants; however, emerging evidence suggests that specialization occurs at the molecular level. To address how the mechanotransduction complex varies in the inner ear, we characterized the expression of paralogous genes that encode components required for mechanotransduction in zebrafish hair cells using RNA-FISH and bioinformatic analysis. Our data indicate striking zonal differences in the expression of two components of the mechanotransduction complex which are known to physically interact, the transmembrane channel-like 1 and 2 (tmc1/2) family members and the calcium and integrin binding 2 and 3 (cib2/3) paralogues. tmc1, tmc2b, and cib3 are largely expressed in peripheral or extrastriolar hair cells, whereas tmc2a and cib2 are enriched in central or striolar hair cells. In addition, a gene implicated in deaf-blindness, ush1c, is highly enriched in a subset of extrastriolar hair cells. These results indicate that specific combinations of these components may optimize responses to mechanical stimuli in subtypes of sensory receptors within the inner ear.

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“…Recombinantly-expressed PCDH15 binds to LHFPL5 and CDH23, and their respective protein complexes have been characterized by cryo-EM 9 and x-ray crystallography 21 . Peptides of TMC1 form a complex with CIB2 and CIB3 14,22 , and mutagenesis experiments in mouse hair cells suggest that TMIE directly interacts with TMC1 12 . In accordance with these studies, the recently elucidated structures of the native C. elegans TMC-1 and TMC-2 complexes revealed a dimeric complex that is composed of two copies of TMC-1/2, TMIE, and CIB2/3, the latter of which is known as CALM-1 in C. elegans 23 .…”

Section: Introductionmentioning

confidence: 99%

Single molecule studies of the native hair cell mechanosensory transduction complex

Clark,

Mitra,

Elferich

et al. 2023

Preprint

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Hearing and balance rely on the conversion of a mechanical stimulus into an electrical signal, a process known as mechanosensory transduction (MT). In vertebrates, this process is accomplished by an MT complex that is located in hair cells of the inner ear. While the past three decades of research have identified many subunits that are important for MT and revealed interactions between these subunits, the composition and organization of a functional complex remains unknown. The major challenge associated with studying the MT complex is its extremely low abundance in hair cells; current estimates of MT complex quantity range from 3-60 attomoles per cochlea or utricle, well below the detection limit of most biochemical assays that are used to characterize macromolecular complexes. Here we describe the optimization of two single molecule assays, single molecule pull-down (SiMPull) and single molecule array (SiMoA), to study the composition and quantity of native mouse MT complexes. We demonstrate that these assays are capable of detecting and quantifying low attomoles of the native MT subunits protocadherin-15 (PCDH15) and lipoma HMGIC fusion partner-like protein 5 (LHFPL5). Our results illuminate the stoichiometry of PCDH15- and LHFPL5-containing complexes and establish SiMPull and SiMoA as productive methods for probing the abundance, composition, and arrangement of subunits in the native MT complex.Impact StatementIn the present work, the authors develop and employ single molecule methods to detect, characterize, and quantitate attomole quantities of the hair cell mechanosensory transduction complex.

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

Cited by 4 publications

References 104 publications

An Open-Source Tool for Automated Human-Level Circling Behavior Detection

An Open-Source Tool for Automated Human-Level Circling Behavior Detection

Differential expression of mechanotransduction complex genes in auditory/vestibular hair cells in zebrafish

Single molecule studies of the native hair cell mechanosensory transduction complex

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