LOXHD1 is indispensable for coupling auditory mechanosensitive channels to the site of force transmission

LOXHD1 is indispensable for coupling auditory mechanosensitive channels to the site of force transmission | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Biological Sciences - Article LOXHD1 is indispensable for coupling auditory mechanosensitive channels to the site of force transmission Pei Wang, Katharine K. Miller, Enqi He, Siddhant S. Dhawan, Christopher L. Cunningham, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3752492/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Sep, 2024 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Hearing is initiated in hair cells by the mechanical activation of ion channels in the hair bundle. The hair bundle is formed by stereocilia organized into rows of increasing heights interconnected by tip links, which convey sound-induced forces to stereocilia tips. The auditory mechanosensitive channels are complexes containing at least four protein-subunits – TMC1/2, TMIE, CIB2, and LHFPL5 – and are located at the tips of shorter stereocilia at a yet-undetermined distance from the lower tip link insertion point. While multiple auditory channel subunits appear to interact with the tip link, it remains unknown whether their combined interaction alone can resist the high-frequency mechanical stimulations owing to sound. Here we show that an unanticipated additional element, LOXHD1, is indispensable for maintaining the TMC1 pore-forming channel subunits coupled to the tip link. We demonstrate that LOXHD1 is a unique element of the auditory mechanotransduction complex that selectively affects the localization of TMC1, but not its close developmental paralogue TMC2. Taking advantage of our novel immunogold scanning electron microscopy method for submembranous epitopes (SUB-immunogold-SEM), we demonstrate that TMC1 normally concentrates within 100-nm of the tip link insertion point. In LOXHD1’s absence, TMC1 is instead mislocalized away from this force transmission site. Supporting this finding, we found that LOXHD1 interacts selectively in vitro with TMC1 but not with TMC2 while also binding to channel subunits CIB2 and LHFPL5 and tip-link protein PCDH15. SUB-immunogold-SEM additionally demonstrates that LOXHD1 and TMC1 are physically connected to the lower tip-link complex in situ. Our results show that the TMC1-driven mature channels require LOXHD1 to stay coupled to the tip link and remain functional, but the TMC2-driven developmental channels do not. As both tip links and TMC1 remain present in hair bundles lacking LOXHD1, it opens the possibility to reconnect them and restore hearing for this form of genetic deafness. Biological sciences/Neuroscience/Auditory system/Hair cell Biological sciences/Biological techniques/Imaging/Molecular imaging Biological sciences/Neuroscience/Auditory system/Transduction Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Full Text Additional Declarations There is NO Competing Interest. Supplementary Files ExtendedDataTable1121323.pdf Extended Data Table 1: Sample size and statistics of each figure panel. Suppvideoregresv3.mov Extended Data Movie 1 Extended Data Movie 1: LOXHD1 is connected to the channel complex and the tip link in vivo, related to Figure 2. Demonstration of TMC1-HA puncta scoring method at IHC row 2 stereocilia tips. ExtendedDataFigs.pdf Extended Data Figures ExtendedDataLegends.pdf Cite Share Download PDF Status: Published Journal Publication published 10 Sep, 2024 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3752492","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Biological Sciences - Article","associatedPublications":[],"authors":[{"id":264183754,"identity":"4b74c88c-6e71-47ff-bd04-4971f932e017","order_by":0,"name":"Pei Wang","email":"","orcid":"","institution":"Department of Otolaryngology-Head and Neck Surgery, Stanford University, 240 Pasteur Drive, Stanford, CA, USA","correspondingAuthor":false,"prefix":"","firstName":"Pei","middleName":"","lastName":"Wang","suffix":""},{"id":264183755,"identity":"006378bc-0f0f-4aff-9495-f44766d9bb3d","order_by":1,"name":"Katharine K. Miller","email":"","orcid":"","institution":"Department of Otolaryngology-Head and Neck Surgery, Stanford University, 240 Pasteur Drive, Stanford, CA, USA","correspondingAuthor":false,"prefix":"","firstName":"Katharine","middleName":"K.","lastName":"Miller","suffix":""},{"id":264183756,"identity":"8b6a2897-01f3-4f24-8ead-42fc53df0021","order_by":2,"name":"Enqi He","email":"","orcid":"","institution":"Department of Otolaryngology-Head and Neck Surgery, Stanford University, 240 Pasteur Drive, Stanford, CA, USA","correspondingAuthor":false,"prefix":"","firstName":"Enqi","middleName":"","lastName":"He","suffix":""},{"id":264183757,"identity":"ac89956f-463d-433c-940e-8e906ab3c616","order_by":3,"name":"Siddhant S. Dhawan","email":"","orcid":"","institution":"Department of Otolaryngology-Head and Neck Surgery, Stanford University, 240 Pasteur Drive, Stanford, CA, USA","correspondingAuthor":false,"prefix":"","firstName":"Siddhant","middleName":"S.","lastName":"Dhawan","suffix":""},{"id":264183758,"identity":"f6652709-39fe-4198-98c1-7bf31cd96010","order_by":4,"name":"Christopher L. Cunningham","email":"","orcid":"","institution":"Pittsburgh Hearing Research Center, Department of Otolaryngology, University of Pittsburgh, Pittsburgh, PA 15213, USA","correspondingAuthor":false,"prefix":"","firstName":"Christopher","middleName":"L.","lastName":"Cunningham","suffix":""},{"id":264183753,"identity":"eb6bfbdd-50ba-4080-883c-51f82982899d","order_by":5,"name":"Nicolas Grillet","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIie3PsQrCMBCA4YNApmjWlL7EQaFSLD5LINBVRycRBFfXCr6C75BS0LFroYvFwcVBEERw0EY3h7SjYP7pEvJxBMDl+tH0YQLA32NsjgCilUgE4c1JMyYdCRiCuisZ8G3dbIlnQVFk14mMx31NsopZSJSesCGJCEtF/FQm0VpTNbQRLHfmL3lDCPi9e46oWeh3IE8RrHLyYNIQfrOTYmmIFgiK+h/CaMsWaojy0lKFQyYT9HIaRBvrlh25XKYjzlfZsWIyxv5+UZdnCwEhv2+I7bmJ67YXLpfL9fe9AN9kTNCTxU+oAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-8007-9246","institution":"Department of Otolaryngology-Head and Neck Surgery, Stanford University, 240 Pasteur Drive, Stanford, CA, USA","correspondingAuthor":true,"prefix":"","firstName":"Nicolas","middleName":"","lastName":"Grillet","suffix":""}],"badges":[],"createdAt":"2023-12-14 09:00:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3752492/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3752492/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-024-51850-4","type":"published","date":"2024-09-10T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":49061598,"identity":"7ff627a2-62dd-4819-acd6-414e01d45c21","added_by":"auto","created_at":"2024-01-02 14:17:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2561875,"visible":true,"origin":"","legend":"\u003cp\u003eThe absence of LOXHD1 leads to hair bundle mechanotransduction deficits and \u0026nbsp;localizes at transducing stereocilia row 2 tips. \u0026nbsp;a, Illustration of a cochlear sensory epithelium, which contains IHCs and OHCs that sense \u0026nbsp;sound-induced forces with their apical hair bundles. b, Illustration of a P21 IHC hair bundle \u0026nbsp;comprising three stereocilia rows interconnected by tip links. c, Cartoon representing the known \u0026nbsp;auditory MET channel complex subunits located near the lower tip-link insertion point. d, The Loxhd1Delta allele contains a large-scale genomic deletion at the Loxhd1 locus (see also Extended \u0026nbsp;Data Fig.1). e, ABR and f, DPOAE thresholds of P21 Loxhd1Delta mice across a 4–45 kHz \u0026nbsp;frequency range. Data are represented as mean ± SEM (Loxhd1+/+, n = 4; Loxhd1Delta/+, n = 6; \u0026nbsp;Loxhd1Delta/Delta, n = 5. Two-way ANOVA (frequency/genotype) followed by the Dunnett \u0026nbsp;multiple comparisons test, with WT means being the reference. Loxhd1Delta/Delta mice values are \u0026nbsp;significantly different from Loxhd1+/+. For all tests in this paper, ns = P \u0026gt; 0.05; * = P≤ 0.05; ** = \u0026nbsp;P ≤ 0.01; *** = P ≤ 0.001; **** = P ≤ 0.0001. Loxhd1Delta/Delta thresholds are significantly \u0026nbsp;different from Loxhd1+/+ between 5.7–45 kHz for ABR and 8–45 kHz for DPOAE (see Extended \u0026nbsp;Data Table 1 for group numbers and P values. Only comparisons between Loxhd1+/+ and \u0026nbsp;Loxhd1Delta/+ are indicated on the graph. g, SEM of apical IHC Loxhd1Delta mice at P11, P21, and \u0026nbsp;P60, with each stereocilia row being colored in post-production. Scale bars = 3 μm. h-k, \u0026nbsp;Mechanotransduction current (I-MET) traces from P7 (h) and P11 (j) IHC Loxhd1+/+ and \u0026nbsp;Loxhd1Delta/Delta mechanically stimulated with a fluid jet and the maximum I-MET currents \u0026nbsp;recorded (i and k). P7: Loxhd1+/+: nmice = 7, ncells = 11; Loxhd1Delta/Delta nmice = 10, ncells = 10; P11: \u0026nbsp;Loxhd1+/+: nmice = 6, ncells = 9; Loxhd1Delta/Delta nmice = 9, ncells = 11. Unpaired two-tailed t test. \u0026nbsp;DV = driving voltage of the fluid jet piezoelectric stimulator. l-m, Hair bundle fluorescent \u0026nbsp;labeling the actin-rich stereocilia, and with an anti-HA antibody to detect LOXHD1 in \u0026nbsp;Loxhd1HA/HA animals. Specific HA staining is detected in Loxhd1HA/HA apical IHC and OHC HBs \u0026nbsp;at P7 (l), P11 and P21 (m) in but not in negative controls without HA. Scale bars = 4 μm. \u0026nbsp;LOXHD1-HA can clearly be detected at the tip of row 2 stereocilia (arrowheads in insets) of \u0026nbsp;apical P11 and P21 IHCs. White scale bars = 4 μm, orange scale bars = 0.4 μm.\u003c/p\u003e","description":"","filename":"FiguresetDEC1320231.png","url":"https://assets-eu.researchsquare.com/files/rs-3752492/v1/823826c949c6e26fae03f65b.png"},{"id":49060785,"identity":"aa235696-9d07-409f-a1ff-11fbb398f162","added_by":"auto","created_at":"2024-01-02 14:09:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2387562,"visible":true,"origin":"","legend":"\u003cp\u003eTMC1 is progressively missing from row 2 stereocilia ti 574 ps of IHCs in the absence \u0026nbsp;of LOXHD1. \u0026nbsp;a, TMC1 immunofluorescence localization (anti-HA on Tmc1HA/HA animals) at the tips of row 2 \u0026nbsp;IHC stereocilia at P7, P11, and P21 showed a progressive reduction in Loxhd1Delta/Delta but not in \u0026nbsp;Loxhd1Delta/+. Insets show high magnification at some row 2 tips. Scale bars = 4 μm. b, \u0026nbsp;Percentage of row 2 tips with TMC1 puncta identified from 3D-volumes per genotype (see \u0026nbsp;Extended Data Fig. 2 and the Material and Methods section for more details). Per group: nmice ≥ \u0026nbsp;3, ncells ≥ 9. One-way ANOVA followed by the Tukey multiple comparisons test (see Extended \u0026nbsp;Data Table 1 for group numbers and P values for all panels). c, TMC2 immunofluorescence \u0026nbsp;localization (anti-MYC on Tmc2MYC/MYC animals) at the tips of row 2 IHC stereocilia at P7 and \u0026nbsp;P11. Scale bars = 4 μm. d, The percentage of row 2 tips with TMC2 puncta was not affected in \u0026nbsp;Loxhd1Delta/Delta at P7 and P11. Per group: nmice ≥ 3, ncells ≥ 9. One-way ANOVA followed by the \u0026nbsp;Tukey multiple comparisons test. e, TMIE immunofluorescence localization (anti-HA on \u0026nbsp;TmieHA/HA animals) at the tips of row 2 IHC stereocilia at P11. Scale bars = 4 μm. f, The \u0026nbsp;percentage of row 2 tips with TMIE puncta was reduced in Loxhd1Delta/Delta at P11 but to a lesser \u0026nbsp;extent than TMC1. Per group: nmice ≥ 3, ncells ≥ 9. Unpaired two-tailed t test.\u003c/p\u003e","description":"","filename":"FiguresetDEC1320232.png","url":"https://assets-eu.researchsquare.com/files/rs-3752492/v1/31ba32bc4608838df2263d23.png"},{"id":49060781,"identity":"4a180801-1c61-45d2-9938-73aa64f01a57","added_by":"auto","created_at":"2024-01-02 14:09:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1198138,"visible":true,"origin":"","legend":"\u003cp\u003eTMC1 is progressively mislocalized away from the first 100 nm of row 2 \u0026nbsp;stereocilia tips of IHCs in the absence of LOXHD1. \u0026nbsp;a, Sub-membranous (or SUB) immunogold-SEM anti-HA with secondary antibody conjugated \u0026nbsp;to 10 nm gold-beads localizes TMC1 at the tip of rows 2 and 3 stereocilia of P21 IHCs. Note that \u0026nbsp;some row 3 stereocilia are disconnected from row 2, while gold-beads are attached to row 2 \u0026nbsp;shafts at the presumptive upper tip link insertion position. Scale bar = 200 nm. b, Distance between TMC1-gold beads to the stereocilia tip in rows 2 and 3 stereocilia measured from SUB immunogold-SEM micrographs in Loxhd1+/+ IHCs. TMC1-gold beads are concentrated within \u0026nbsp;100 nm from the stereocilia tips (see the focused panel). Medians are indicated by a blue line. \u0026nbsp;P21: nmice = 1; ncells = 13; row 2: nstereocilia = 40; ngold = 62; row 3: nstereocilia = 45; ngold = 77. c-e, \u0026nbsp;SUB-immunogold-SEM anti-HA detects TMC1-associated gold beads (arrowheads) at IHC row \u0026nbsp;2 tips of Loxhd1+/+ and Loxhd1Delta/Delta at P7, P11, and P21. Scale bar = 200 nm. f, Distance \u0026nbsp;between TMC1-gold beads to the stereocilia tip in row 2 stereocilia measured from SUB immunogold-SEM micrographs in Loxhd1+/+ and Loxhd1Delta/Delta IHCs. Per group: P7: nmice ≥ 2; \u0026nbsp;ncells ≥ 18; nstereocilia ≥ 113; ngold =168 for Loxhd1+/+ and 194 for Loxhd1Delta/Delta; P11: nmice = 2; \u0026nbsp;ncells ≥ 16; nstereocilia ≥ 51; ngold ≥ 62; ngold 204 for Loxhd1+/+ and 62 for Loxhd1Delta/Delta; P21: nmice \u0026nbsp;≥ 1; ncells ≥ 13; nstereocilia ≥ 30; ngold = 62 for Loxhd1+/+ and 37 for Loxhd1Delta/Delta (see Extended \u0026nbsp;Data Table 1). Medians are indicated by a blue line. g, Distance between TMC1-gold beads to \u0026nbsp;the stereocilia tip in row 3 stereocilia measured from SUB-immunogold-SEM micrographs in \u0026nbsp;Loxhd1+/+ and Loxhd1Delta/Delta IHCs. Per group: P11: nmice = 2; ncells ≥ 14; nstereocilia = 117 for \u0026nbsp;Loxhd1+/+ and 12 for Loxhd1Delta/Delta; ngold = 135 for Loxhd1+/+ and 62 for Loxhd1Delta/Delta; per \u0026nbsp;group: P21: nmice ≥ 1; ncells ≥ 13; nstereocilia = 45 for Loxhd1+/+ and 0 for Loxhd1Delta/Delta; ngold = 62 \u0026nbsp;for Loxhd1+/+ and 0 for Loxhd1Delta/Delta (see Extended Data Table 1). h-i, Stereocilia \u0026nbsp;classification based on the TMC1-gold distribution pattern (present in the stereocilia, with at \u0026nbsp;least one gold bead in the first 100 nm, with at least one gold bead below 100 nm, and without \u0026nbsp;any gold beads). Row 2 (h): per group: P7: nmice ≥ 1; ncells ≥ 16; nstereocilia ≥ 166; P11-P21: nmice ≥ \u0026nbsp;1; ncells ≥ 13; nstereocilia ≥ 130; row 3 (i): per group: P11: nmice ≥ 1; ncells ≥ 13; nstereocilia ≥ 148; P21: \u0026nbsp;nmice = 1; ncells ≥ 11; nstereocilia ≥ 65 (see Extended Data Table 1).\u003c/p\u003e","description":"","filename":"FiguresetDEC1320233.png","url":"https://assets-eu.researchsquare.com/files/rs-3752492/v1/46e9f480d9cef9722406b3c5.png"},{"id":49060783,"identity":"896a685f-7065-4534-a730-b188b57d6c6a","added_by":"auto","created_at":"2024-01-02 14:09:29","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1967492,"visible":true,"origin":"","legend":"\u003cp\u003eStereocilia Ca2+ entry zones, probed 620 with BAIAP2L2, are relocated from tips to \u0026nbsp;shaft. \u0026nbsp;a, Anti-BAIAP2L2 immunofluorescence on P21 apical HCs shows a strong signal at transducing \u0026nbsp;stereocilia tips. In the mechanotransduction deficient IHC mutants TmieKO/KO and Tmc1KO/KO, \u0026nbsp;BAIAP2L2 expression is very sparse and not enriched at any stereocilia location. In \u0026nbsp;Loxhd1Delta/Delta, a strong BAIAP2L2 signal is detected in row 2 stereocilia but with a different \u0026nbsp;distribution than control Loxhd1Delta/+ littermates. Boxes correspond to magnified panels. Scale \u0026nbsp;bars = 2 μm. b, Stereocilia classification based on the BAIAP2L2 fluorescence signal \u0026nbsp;distribution pattern: only at the tip, at the tip and along the shaft, at the shaft, or absent from \u0026nbsp;stereocilia. Data are represented as mean ± SEM. Per group: nmice ≥ 2; ncells ≥ 8; Mann–Whitney \u0026nbsp;test, two-tailed (see Extended Data Table 1). c, SUB-immunogold-SEM anti-BAIAP2L2 shows \u0026nbsp;the enrichment of gold beads at the tips of rows 2 and 3 P21 IHC stereocilia in Loxhd1+/+, while \u0026nbsp;gold beads are more distant from the tip in Loxhd1Delta/Delta. Arrowheads indicate the gold beads \u0026nbsp;closest to the tips. Scale bars = 500 nm. d-e, Distance between BAIAP2L2-gold beads to tips of \u0026nbsp;rows 2 (d) and 3 (e) stereocilia measured from SUB-immunogold-SEM micrographs in \u0026nbsp;Loxhd1+/+ and Loxhd1Delta/Delta P21. IHCs corresponded to ~70% apical, ~12% medial, and ~18% \u0026nbsp;basal. Per group: P21 row 2 (d) nmice ≥ 2; ncells ≥ 34; nstereocilia ≥ 168; ngold ≥ 438; row 3 (e) nmice ≥ \u0026nbsp;2; ncells ≥ 34; nstereocilia = 155 for Loxhd1+/+ and 4 for Loxhd1Delta/Delta; ngold = 303 for Loxhd1+/+ \u0026nbsp;and 4 for Loxhd1Delta/Delta (see Extended Data Table 1). f-g, Stereocilia classification based on the \u0026nbsp;BAIAP2L2-gold distribution pattern: present in the stereocilia, with at least one gold bead in the \u0026nbsp;first 100 nm, with at least one gold bead below 100 nm, and without any gold beads. Row 2 (f): \u0026nbsp;per group: nmice ≥ 2; ncells ≥ 33; nstereocilia ≥ 289; row 3 (g): per group: nmice ≥ 2; ncells = 32; nstereocilia \u0026nbsp;= 280 for Loxhd1+/+ and 70 for Loxhd1Delta/Delta (see Extended Data Table 1).\u003c/p\u003e","description":"","filename":"FiguresetDEC1320234.png","url":"https://assets-eu.researchsquare.com/files/rs-3752492/v1/81a418c5d7d8383fa323d218.png"},{"id":49060784,"identity":"b807dfdd-d398-4fba-8db3-d04731b704d4","added_by":"auto","created_at":"2024-01-02 14:09:29","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1272942,"visible":true,"origin":"","legend":"\u003cp\u003eLOXHD1 interacts in vitro the channel complex and the tip link proteins and is \u0026nbsp;connected to them in vivo. \u0026nbsp;a-d, Co-immunoprecipitation experiments from HEK293t cells overexpressing LOXHD1-HA \u0026nbsp;with tagged channel complex or tip link proteins. FLAG-TMC1 is co-immunoprecipitated with \u0026nbsp;LOXHD1-HA using anti-HA magnetic beads, but FLAG-TMC2 is not (a); CIB2-V5 (b), FLAG LHFPL5 (c) and PCDH15 (d) were co-immunoprecipitated with LOXHD1-HA, while TMIE FLAG was not (b). Each experiment was replicated at least thrice; (a) was replicated five times. \u0026nbsp;Predicted protein sizes based on primary sequence: LOXHD1-HA: 237 kDa; TMC1-FLAG: 88 \u0026nbsp;kDa; TMC2-FLAG: 104 kDa; CIB2-V5: 23 kDa; TMIE-FLAG: 18 kDa; FLAG-LHFPL5: 25 \u0026nbsp;kDa; PCDH15: 216 kDa. IP: Immunoprecipitation. e, In SUB experiments on P21 IHCs, some \u0026nbsp;row 2 tips are detached from the rest of the stereocilia but still connected to the tip links \u0026nbsp;(arrowheads and high magnification). Anti-HA gold-beads were specifically found at detached \u0026nbsp;row 2 tips for LOXHD1-HA and TMC1-HA but not in the WT sample. The lower panels are \u0026nbsp;high magnification of detached row 2 tips. Scale bars: 500 nm for the low mag pictures, 50 nm \u0026nbsp;for the high mag ones. f, Quantification of the percentage of P21 IHC detached row 2 tips \u0026nbsp;containing gold. Loxhd1HA/HA anti HA: nmice = 2, ncells = 17, ndetached tip with gold / total = 31/54; WT \u0026nbsp;anti-HA: nmice = 1, ncells = 5, ndetached tip with gold / total = 0/11; Tmc1HA/HA anti HA: nmice = 2, ncells = 10, \u0026nbsp;ndetached tip with gold / total = 34/44; WT anti-BAIAP2L2: nmice = 2, ncells = 14, ndetached tip with gold / total = \u0026nbsp;1/42. g, Cartoon summarizing interactions between LOXHD1 and the MET channel complex and \u0026nbsp;tip link. Note that LOXHD1 does not interact with TMIE.\u003c/p\u003e","description":"","filename":"FiguresetDEC1320235.png","url":"https://assets-eu.researchsquare.com/files/rs-3752492/v1/d4bb50e5929538b8f0114933.png"},{"id":64278844,"identity":"4e3b9817-f610-47de-8172-5ad59da204e2","added_by":"auto","created_at":"2024-09-11 07:12:10","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1913932,"visible":true,"origin":"","legend":"Article File","description":"","filename":"MANUSCRIPTDEC132023.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3752492/v1_covered_2c1aa22b-aa5c-43da-a78a-6f53bac2ca7f.pdf"},{"id":49060780,"identity":"da3b29a5-d658-4835-9d8d-d9d3f8e8cf38","added_by":"auto","created_at":"2024-01-02 14:09:29","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":120617,"visible":true,"origin":"","legend":"\u003cp\u003eExtended Data Table 1: Sample size and statistics of each figure panel.\u003c/p\u003e","description":"","filename":"ExtendedDataTable1121323.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3752492/v1/021a252f1f7db46b6d800f79.pdf"},{"id":49060788,"identity":"605a534b-beb3-4029-bdef-cb5d030c6440","added_by":"auto","created_at":"2024-01-02 14:09:31","extension":"mov","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":57238904,"visible":true,"origin":"","legend":"\u003cp\u003eExtended Data Movie 1\u003c/p\u003e\n\u003cp\u003eExtended Data Movie 1: LOXHD1 is connected to the channel complex and the tip link in vivo, related to Figure 2. Demonstration of TMC1-HA puncta scoring method at IHC row 2 stereocilia tips.\u003c/p\u003e","description":"","filename":"Suppvideoregresv3.mov","url":"https://assets-eu.researchsquare.com/files/rs-3752492/v1/db461f44a01219f7ba658ecd.mov"},{"id":49060787,"identity":"8f7015b4-0bf1-4f9d-ae29-23ba946d71bc","added_by":"auto","created_at":"2024-01-02 14:09:30","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":4607828,"visible":true,"origin":"","legend":"\u003cp\u003eExtended Data Figures\u003c/p\u003e","description":"","filename":"ExtendedDataFigs.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3752492/v1/03e01d9e3994177933e0c358.pdf"},{"id":49060782,"identity":"29ad214b-b3ff-401e-9bb4-41bd12c178fd","added_by":"auto","created_at":"2024-01-02 14:09:29","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":202982,"visible":true,"origin":"","legend":"","description":"","filename":"ExtendedDataLegends.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3752492/v1/1214f14ba1dc0ced9d77e46e.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"LOXHD1 is indispensable for coupling auditory mechanosensitive channels to the site of force transmission","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3752492/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3752492/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Hearing is initiated in hair cells by the mechanical activation of ion channels in the hair bundle. The hair bundle is formed by stereocilia organized into rows of increasing heights interconnected by tip links, which convey sound-induced forces to stereocilia tips. The auditory mechanosensitive channels are complexes containing at least four protein-subunits – TMC1/2, TMIE, CIB2, and LHFPL5 – and are located at the tips of shorter stereocilia at a yet-undetermined distance from the lower tip link insertion point. While multiple auditory channel subunits appear to interact with the tip link, it remains unknown whether their combined interaction alone can resist the high-frequency mechanical stimulations owing to sound. Here we show that an unanticipated additional element, LOXHD1, is indispensable for maintaining the TMC1 pore-forming channel subunits coupled to the tip link. We demonstrate that LOXHD1 is a unique element of the auditory mechanotransduction complex that selectively affects the localization of TMC1, but not its close developmental paralogue TMC2. Taking advantage of our novel immunogold scanning electron microscopy method for submembranous epitopes (SUB-immunogold-SEM), we demonstrate that TMC1 normally concentrates within 100-nm of the tip link insertion point. In LOXHD1’s absence, TMC1 is instead mislocalized away from this force transmission site. Supporting this finding, we found that LOXHD1 interacts selectively in vitro with TMC1 but not with TMC2 while also binding to channel subunits CIB2 and LHFPL5 and tip-link protein PCDH15. SUB-immunogold-SEM additionally demonstrates that LOXHD1 and TMC1 are physically connected to the lower tip-link complex in situ. Our results show that the TMC1-driven mature channels require LOXHD1 to stay coupled to the tip link and remain functional, but the TMC2-driven developmental channels do not. As both tip links and TMC1 remain present in hair bundles lacking LOXHD1, it opens the possibility to reconnect them and restore hearing for this form of genetic deafness.","manuscriptTitle":"LOXHD1 is indispensable for coupling auditory mechanosensitive channels to the site of force transmission","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-02 14:09:25","doi":"10.21203/rs.3.rs-3752492/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"80b21a37-0af5-47ba-9479-56892c974cb7","owner":[],"postedDate":"January 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":27853002,"name":"Biological sciences/Neuroscience/Auditory system/Hair cell"},{"id":27853003,"name":"Biological sciences/Biological techniques/Imaging/Molecular imaging"},{"id":27853004,"name":"Biological sciences/Neuroscience/Auditory system/Transduction"}],"tags":[],"updatedAt":"2024-09-11T07:11:57+00:00","versionOfRecord":{"articleIdentity":"rs-3752492","link":"https://doi.org/10.1038/s41467-024-51850-4","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2024-09-10 04:00:00","publishedOnDateReadable":"September 10th, 2024"},"versionCreatedAt":"2024-01-02 14:09:25","video":"","vorDoi":"10.1038/s41467-024-51850-4","vorDoiUrl":"https://doi.org/10.1038/s41467-024-51850-4","workflowStages":[]},"version":"v1","identity":"rs-3752492","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3752492","identity":"rs-3752492","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}