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

总结 我们的长期目标是在分子水平上了解毛细胞机械传导是如何工作的，以及为什么它会发生。 不同类型的听力损失。我们的策略是研究导致人类听力损失的基因， 在老鼠身上做模型。这项研究的一个关键方面依赖于定位耳聋蛋白在细胞和 内耳的亚细胞水平。在毛细胞的机械感觉器中，被称为毛束，高- 蛋白质的分辨率定位为确定其功能提供了关键信息。比如说 机械转换机构本身沿尖端连接端沿着不对称地组织。头端链接 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， 命令关闭通道。因此，定位于上尖端-连接插入复合物的蛋白质表明， 在头端连杆张力维持中的潜在作用。与此相反，位于较低尖端连接插入位点的蛋白质 表明与机械-电换能器（MET）通道复合体的作用。 高分辨率蛋白定位通常通过免疫金标记样品来实现， 随后包埋、切片并使用透射电子显微镜成像。但这 这种方法非常耗时，不适合广泛的量化。另一种方法 使用扫描电子显微镜（SEM）对免疫金标记的样品的表面进行成像，然而， 方法仅限于研究细胞外蛋白。由于毛束的许多蛋白质是内部的， 在膜附近，我们假设免疫金扫描电镜可以适用于定位这些 内部蛋白质这样的技术改进将允许在纳米尺度上定位蛋白质分布 从大量的毛细胞，导致增加的样本数量更强的量化。 在本提案的具体目标1中，我们将最终确定我们的免疫金-SEM方案， 定位于静纤毛膜下的内部蛋白质或静纤毛膜的整体蛋白质 膜的我们的初步数据表明，内部肌动蛋白结合蛋白，EPS 8，保持其 在最高的静纤毛的尖端特征性富集。同时，毛束结构及其 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PMCA 2a。 在具体目标2中，我们将利用这些免疫金-SEM协议来定位 纳米级分辨率的机械传导通道。TMC 1、TMC 2的蛋白质分布 （参与孔）和TMIE（TMCs活性所需）将被映射在转导的尖端。 静纤毛MET通道位置，预计位于TMC和TMIE之间的交叉点 分布，并测量其到尖端链接的距离。总的来说，我们希望不仅提供第一个 MET通道组件的纳米级地图，但也是一种对许多其他有价值的开创性技术 研究领域，如细胞粘附，平面细胞极性，纤毛病，轴突导向等。

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.