LOXHD1 is indispensable for maintaining TMC1 auditory mechanosensitive channels at the site of force transmission

Hearing begins in hair cells with the mechanical activation of ion channels in the hair bundle. This bundle consists of stereocilia arranged in rows of increasing heights, connected by tip links that transmit sound-induced forces to shorter stereocilia tips. Auditory mechanotransduction channel complexes, composed of proteins TMC1/2, TMIE, CIB2, and LHFPL5, are located at the tips of shorter stereocilia. While most components can interact with the tip link in vitro, their ability to maintain the channel complexes at the tip link in vivo is uncertain. Here we show that an additional component, LOXHD1, is essential for keeping TMC1-pore forming subunits at the tip link but is dispensable for TMC2. Using SUB-immunogold-SEM, we showed that TMC1 localizes near the tip link but mislocalizes without LOXHD1. LOXHD1 selectively interacts with TMC1, CIB2, LHFPL5, and tip-link protein PCDH15. Our results demonstrate that TMC1-driven mature auditory channels require LOXHD1 to stay connected to the ..., Scanning electron microscopy (SEM) SEM sample preparation and imaging were conducted as follows: The inner ears were isolated in dissection buffer (DB) (1 X HBSS with Ca2+ and Mg2+, with osmolarity adjusted to 310 mOsm with D-glucose) and fixed in 4% PFA diluted from 32% stock in this buffer for 30 min at RT. The inner ears were then dissected to remove bone structures, the stria vascularis, and Reissner’s and tectorial membranes. The samples were refixed in 2.5% glutaraldehyde and 4% PFA in DB overnight at 4 °C, washed, dehydrated in ethanol (30%, 50%, 75%, 95%, 100%, and 100%, 5 min incubations), and processed to the critical drying point using Autosamdri-815A (Tousimis). The cochleae were mounted on studs using silver paint and coated with 4-nm palladium (sputter coater EMS150TS, Electron Microscopy Sciences). The samples were imaged with a 5-kV accelerated voltage and a 13-pA beam current using a secondary electron detector on an FEI Magellan 400 XHR Field Emission Scanning Electron..., , # Raw data of the publication: \"LOXHD1 is indispensable for maintaining TMC1 auditory mechanosensitive channels at the site of force transmission. \[Raw data of the publication: \"LOXHD1 is indispensable for maintaining TMC1 auditory mechanosensitive channels at the site of force transmission.\"]([https://doi.org/10.5061/dryad.v15dv4256](https://doi.org/10.5061/dryad.v15dv4256)) This dataset includes raw scanning electron microscopy (SEM) images, airyscan immunofluorescent (IF) microscopy images, and SUB-Immunogold-SEM images acquired and analyzed for Figures of the publication: \"LOXHD1 is indispensable for maintaining TMC1 auditory mechanosensitive channels at the site of force transmission.\" #### Description of the data and file structure The data are organized in folders following the names of the Figures in the publication. Subfolders are used for better data organization based on ages, genotypes, and ID of independent experiments. Information on animal ages, genotypes, labeling, ...听觉始于毛细胞（hair cells）中毛束（hair bundle）内离子通道的机械激活。该毛束由高度依次递增的静纤毛（stereocilia）阵列组成，静纤毛间通过顶端连接丝（tip links）相连，后者可将声音诱导的力传递至较短静纤毛的尖端。由TMC1/2、TMIE、CIB2及LHFPL5蛋白组成的听觉机械转导通道复合物，定位于较短静纤毛的尖端。尽管多数复合物组分可在体外（in vitro）与顶端连接丝相互作用，但它们在体内（in vivo）维持顶端连接丝处通道复合物的能力尚不明确。本研究发现，额外组分LOXHD1对于将孔形成亚基TMC1保留在顶端连接丝处是必需的，但对TMC2则非必需。通过SUB-免疫金扫描电镜（SUB-immunogold-SEM），我们证实TMC1定位于顶端连接丝附近，而在缺失LOXHD1时则会出现定位异常。LOXHD1可与TMC1、CIB2、LHFPL5及顶端连接丝蛋白PCDH15发生选择性相互作用。本研究结果表明，由TMC1介导的成熟听觉通道需要LOXHD1以维持与……的连接，扫描电子显微镜（SEM） SEM样品制备与成像流程如下：在解剖缓冲液（DB，即含Ca²+和Mg²+的1×HBSS（Hank平衡盐溶液），并用D-葡萄糖将渗透压调节至310 mOsm）中分离内耳，随后用32%多聚甲醛（PFA）原液稀释至4%的该缓冲液固定样本30 min（室温，RT）。随后解剖内耳，去除骨结构、血管纹（stria vascularis）、Reissner膜（Reissner’s membrane）及盖膜（tectorial membrane）。将样本在4℃下用含2.5%戊二醛（glutaraldehyde）和4% PFA的DB缓冲液再次固定过夜，清洗后用乙醇进行梯度脱水（30%、50%、75%、95%、100%、100%，每次孵育5 min），并使用Autosamdri-815A（Tousimis）进行临界点干燥处理。用导电银胶将耳蜗固定于样品台，随后使用EMS150TS离子溅射镀膜仪（Electron Microscopy Sciences）溅射4 nm厚的钯层。使用FEI Magellan 400 XHR场发射扫描电子显微镜的二次电子检测器，以5 kV加速电压、13 pA束流对样本进行成像…… # 本研究论文的原始数据："LOXHD1是维持TMC1听觉机械敏感通道于力传递位点的必需蛋白" [本研究论文的原始数据："LOXHD1是维持TMC1听觉机械敏感通道于力传递位点的必需蛋白"](https://doi.org/10.5061/dryad.v15dv4256) 本数据集包含为该论文《LOXHD1是维持TMC1听觉机械敏感通道于力传递位点的必需蛋白》的配图所获取和分析的原始扫描电子显微镜（SEM）图像、Airyscan免疫荧光（IF）显微镜图像及SUB-免疫金扫描电镜（SUB-Immunogold-SEM）图像。 #### 数据与文件结构说明 数据按照论文中配图的名称进行文件夹分类。为实现更优的数据组织，将根据动物年龄、基因型（genotype）及独立实验ID设置子文件夹。相关信息包括动物年龄、基因型、标记方式……