Molecular Basis of Hair Cell Stereocilia Bundle Morphology

摘要-摘要 使用一个新的蛋白质数据库，在出生后不久，小鼠内耳感觉毛细胞中活跃合成， 我们已经鉴定了一种新的听力损失蛋白，多囊肾和肝病1-样1（PKHD 1 L1）， 以前未显示参与毛细胞功能，或定位于毛细胞静纤毛束。通过特异性 从小鼠内耳感觉毛细胞中去除PKHD 1 L1，我们发现PKHD 1 L1的缺失导致 听力损失为了了解PKHD 1 L1缺陷小鼠发生听力损失的机制，三个密切相关的研究表明， 为这项研究提出了两个目标。由于PKHD 1 L1以前没有被证明参与头发 细胞功能，或已定位于毛细胞静纤毛束，在目的1中，我们将PKHD 1 L1定位于表面 小鼠毛细胞静纤毛在束成熟期间（即出生后的前三周内），使用高度 精确的电子显微镜蛋白定位技术。我们初步的免疫金抗体标记 结果在出生后第4天PKHD 1 L1定位于静纤毛的尖端。在目的2中，我们测试PKHD 1 L1的功能 通过评估毛细胞特异性PKHD 1 L1基因敲除小鼠的听力缺陷。我们发现，“静纤毛 PKHD 1 L1缺陷型静纤毛的顶端缺少“外套”。我们现在将特别关注 PKHD 1 L1-缺陷对覆膜附着冠、维管束内聚、维管束发育的影响 刺激时的运动以及毛细胞转导电流的性质。通过研究PKHD 1 L1- 缺乏小鼠线没有'外套'在尖端的静纤毛，我们提出了一个干净的实验范例， 测试PKHD 1 L1（因此，涂层）对束内聚性和滑动粘附性的贡献。最后，在目标3中， 基于PKHD 1 L1的预测结构域结构，表明可能是同源的（即与其自身）， 异聚体（与其他蛋白质）相互作用，我们将进行蛋白质相互作用实验，以揭示可能的 与其他蛋白质的相互作用参与形成盖膜或其附着到静纤毛，如 Stereocilin、CEACAM 16和tectorin。

Summary – Abstract Using a novel database of proteins actively synthesized in mouse inner ear sensory hair cells shortly after birth, we have identified a novel hearing loss protein, Polycystic Kidney and Hepatic Disease 1-Like 1 (PKHD1L1), previously not shown to participate in hair cell function, or localized to hair cell stereocilia bundles. By specifically removing PKHD1L1 from mouse inner ear sensory hair cells, we show that absence of PKHD1L1 results in hearing loss. In order to understand the mechanism by which PKHD1L1-deficient mice develop hearing loss, three closely coupled aims are proposed for this study. Since PKHD1L1 has not been previously shown to participate in hair cell function, or been localized to hair cell stereocilia bundles, in Aim 1 we will localize PKHD1L1 on the surface of mouse hair cell stereocilia during bundle maturation (i.e. within the first three weeks after birth), using highly precise electron microscopy protein localization techniques. Our preliminary immunogold antibody labeling results localize PKHD1L1 to the tips of stereocilia at postnatal day 4. In Aim 2 we test the function of PKHD1L1 by evaluating the hearing deficit of the hair cell-specific PKHD1L1 knock-out mouse. We show that ‘stereocilia coat’ is absent from the tips of PKHD1L1-deficient stereocilia. We will now specifically focus on the implications of PKHD1L1-deficiency on development of tectorial membrane attachment crowns, bundle cohesion, bundle motion upon stimulation, and the properties of the hair cell transduction current. By studying a PKHD1L1- deficient mouse line without the ‘coat’ at the tips of stereocilia, we propose a clean experimental paradigm to test PKHD1L1’s (hence, the coat’s) contribution to bundle cohesion, and sliding adhesion. Lastly, in our Aim 3, based on the predicted domain structure of PKHD1L1 suggesting possible homomeric (i.e. with itself) and heteromeric (with other proteins) interactions, we will perform protein interaction experiments to reveal possible interactions with other proteins involved in forming the tectorial membrane or its attachment to stereocilia, like stereocilin, CEACAM16 and tectorin.