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

摘要 我们的长期目标是在分子水平上了解毛细胞机械转导是如何工作的，以及为什么 在不同类型的听力损失中失败。我们的策略是通过以下方式研究导致人类听力损失的基因 在老鼠身上对它们进行建模。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 内耳中的亚细胞水平。在毛细胞的机械感觉细胞器中，称为发束，高度- 蛋白质的分辨率定位为确定其功能提供了关键信息。例如， 机械传递机械本身是沿着尖端链节末端不对称地组织的。提示链接 纳米纤维将偏转力从较高的立体纤毛传递到成对的较短的立体纤毛的尖端 命令打开通道门。因此，位于上端-连接插入复合体的蛋白质表明 在顶端连杆张力维持中的潜在作用。相反，定位于较低尖端连接插入位点的蛋白质 建议与机电传感器(MET)通道复合体一起发挥作用。 高分辨率蛋白质定位通常是通过样品的免疫金标记法实现的，这些样品是 随后使用透射电子显微镜对其进行包埋、切片和成像。不过，这个 这种方法非常耗时，不适合进行广泛的量化。另一种方法 使用扫描电子显微镜(SEM)对免疫金标记样的表面进行成像，然而这 方法仅限于研究胞外蛋白。因为发束中的许多蛋白质都是内部的，但在 在膜的附近，我们假设免疫金扫描可以用来定位这些 体内蛋白质。这样的技术改进将允许在纳米尺度上定位蛋白质分布。 从大量的毛细胞中提取，从而增加了样本数量，从而实现了更强的量化。 在这项提案的具体目标1中，我们将最终确定我们的免疫金-扫描电子显微镜方案，致力于 定位于立体纤毛膜下的内部蛋白质或构成立体纤毛的蛋白质 薄膜。我们的初步数据显示，内部肌动蛋白结合蛋白Eps8维持其 在最高的立体纤毛的顶端有特征性的浓集。同时，毛束的结构和它的 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。 在特定的目标2中，我们将利用这些免疫金-扫描电子显微镜技术来定位 纳米级的机械转导通道。TMC1、TMC2的蛋白质分布 (参与毛孔)和TMIE(TMCS活动所需)将被绘制在转换的顶端 立体纤毛。MET通道位置，预计位于TMCS和TMIE之间的交叉口 分布，以及它到尖端链节的距离将被测量。总体而言，我们不仅希望提供第一个 MET通道组件的纳米级地图，但也是一项对许多其他技术有价值的开创性技术 研究领域包括细胞黏附、平面细胞极性、纤毛病变和轴突引导等。

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.