High-resolution localization of the hair cell mechanotransduction channel components by immunogold-scanning electronic microscopy

通过免疫金扫描电子显微镜高分辨率定位毛细胞机械转导通道成分

基本信息

批准号：
10196092
负责人：
Nicolas Grillet
金额：
$ 23.71万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-04-01 至 2023-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10196092
关键词：
Actin-Binding Protein Address Adopted Animals Antibodies Apical Area Binding Cell Adhesion Cell membrane Characteristics Cochlea Communities Complex Consumption Data Dehydration Development Developmental Biology Disease EPS8 gene Electron Microscope Electron Microscopy Epitopes Extracellular Protein Genes Goals Gold Hair Hair Cells Human Image Imaging Techniques In Vitro Integral Membrane Protein Ion Channel Knock-in Mouse Label Labyrinth Link Lipids Location Maintenance Maps Measures Membrane Membrane Proteins Methods Microscopic Microscopy Modeling Molecular Mus Organelles Outer Hair Cells Pathology Physiological Positioning Attribute Preparation Problem Solving Proteins Protocols documentation Research Resolution Role Sampling Scanning Scanning Electron Microscopy Seminal Signal Transduction Site Slice Structure Surface Techniques Testing Time Transducers Work axon guidance base ciliopathy cold temperature deafness experimental study extracellular hair cell regeneration hearing impairment imaging approach mechanotransduction mutant nanofilament nanoscale novel strategies planar cell polarity postnatal preservation protein distribution protein protein interaction research study sample fixation sound tool transmission process

项目摘要

SUMMARY Our long-term goal is to understand at the molecular level how hair cell mechanotransduction works and why it fails in different types of hearing loss. Our strategy is to study genes that cause hearing loss in humans by modeling them in mice. A critical aspect of this research relies on localizing deafness proteins at cellular and subcellular levels in the inner ear. In the hair cell mechanosensory organelle, known as the hair bundle, high- resolution localization of proteins provides critical information for defining their functions. For example, the mechanotransduction machinery itself is organized asymmetrically along the tip link ends. Tip link nanofilaments transmit the deflection force from taller stereocilia to the tips of paired, shorter stereocilia in order to gate the channel. Therefore, a protein localized at the upper tip-link insertion complex indicates a potential role in tip-link tension maintenance. In contrast, a protein localized at the lower tip-link insertion site suggests a role with the mechano-electrical transducer (MET) channel complex. High-resolution protein localization is routinely achieved by immunogold-labeling of samples, which are subsequently embedded, sliced, and imaged using transmission electron microscopes. However, this approach is massively time-consuming and not suited for extensive quantification. An alternative approach images the surface of immunogold-labeled samples using scanning electron microscopy (SEM), however this method is limited to investigating extracellular proteins. As many proteins of the hair bundle are internal but in the vicinity of the membrane, we hypothesized that immunogold-SEM could be adapted to localize these internal proteins. Such a technical improvement would allow localization of protein distribution at the nanoscale from large numbers of hair cells, resulting in increased sample numbers for stronger quantification. In Specific Aim 1 of this proposal, we will finalize our immunogold-SEM protocols dedicated either to localizing internal proteins beneath the stereocilia membrane or proteins integral to the stereociliary membrane. Our preliminary data show that the internal actin-binding protein, EPS8, maintains its characteristic enrichment at the tip of the tallest stereocilia. At the same time, the hair bundle structure and its links are preserved. To test our membrane protein method, we will localize transmembrane protein PMCA2a. In Specific Aim 2, we will capitalize on these immunogold-SEM protocols to locate the mechanotransduction channels at nanoscale resolution. The protein distributions of TMC1, TMC2 (participating in the pore), and TMIE (required for TMCs activity), will be mapped at the tip of transducing stereocilia. The MET channel location, expected to be at the intersection between the TMCs and TMIE distributions, and its distance to the tip link will be measured. Overall, we expect to provide not only the first nanoscale map of the MET channel components, but also a seminal technique valuable to many other research fields such as cell adhesion, planar cell polarity, ciliopathies, and axon guidance among others.

概括我们的长期目标是在分子水平上了解毛细胞机械转导的工作原理及其原因不同类型的听力损失都失败。我们的策略是研究导致人类听力损失的基因在小鼠中对它们进行建模。这项研究的一个关键方面依赖于将耳聋蛋白定位在细胞和内耳的亚细胞水平。在毛细胞机械感觉细胞器（称为发束）中，高蛋白质的分辨率定位为定义其功能提供了关键信息。例如，机械传导机械本身沿着尖端连杆末端不对称地组织。提示链接纳米丝将偏转力从较高的静纤毛传递到成对的、较短的静纤毛的尖端命令来控制通道。因此，位于上部尖端连接插入复合物的蛋白质表明在尖端连杆张力维持中的潜在作用。相反，位于下部尖端连接插入位点的蛋白质表明与机电换能器（MET）通道复合体的作用。高分辨率蛋白质定位通常通过样品的免疫金标记来实现，这些样品是随后使用透射电子显微镜进行嵌入、切片和成像。然而，这该方法非常耗时且不适合广泛量化。另一种方法使用扫描电子显微镜 (SEM) 对免疫金标记样品的表面进行成像，但是这该方法仅限于研究细胞外蛋白质。由于发束的许多蛋白质都在内部，但在在膜附近，我们假设免疫金-SEM 可以适用于定位这些内部蛋白质。这种技术改进将允许蛋白质分布在纳米尺度上定位来自大量毛细胞，导致样本数量增加以进行更强的定量。在本提案的具体目标 1 中，我们将最终确定免疫金-SEM 协议，专用于定位静纤毛膜下方的内部蛋白质或静纤毛不可或缺的蛋白质膜。我们的初步数据表明，内部肌动蛋白结合蛋白 EPS8 保持其最高静纤毛尖端的特征富集。同时，发束结构及其链接被保留。为了测试我们的膜蛋白方法，我们将定位跨膜蛋白 PMCA2a。在具体目标 2 中，我们将利用这些免疫金-SEM 协议来定位纳米级分辨率的力传导通道。 TMC1、TMC2的蛋白分布（参与孔）和 TMIE（TMC 活动所需）将被映射在转导尖端静纤毛。 MET 通道位置，预计位于 TMC 和 TMIE 的交叉点分布，并将测量其到尖端链接的距离。总的来说，我们期望不仅提供第一个 MET 通道成分的纳米级图，也是对许多其他人有价值的开创性技术研究领域包括细胞粘附、平面细胞极性、纤毛病和轴突引导等。