Molecular Basis of Hair Cell Stereocilia Bundle Morphology

摘要-摘要 利用一个新的蛋白质数据库，该数据库包含出生后不久小鼠内耳感觉毛细胞中活跃合成的蛋白质， 我们已经鉴定出一种新的听力损失蛋白，多囊肾和肝病1样蛋白(PKHD1L1)， 以前未显示参与毛细胞功能，或定位于毛细胞立体纤毛束。通过特别指定 从小鼠内耳感觉毛细胞中去除PKHD1L1，我们发现缺少PKHD1L1会导致 听力损失。为了了解PKHD1L1缺陷小鼠发生听力损失的机制，有三个密切相关的研究 本研究提出了耦合目标。因为PKHD1L1以前没有被显示参与头发 细胞功能，或定位于毛细胞立体纤毛束，在目标1中，我们将PKHD1L1定位在表面 在毛束成熟期间(即在出生后的前三周内)小鼠毛细胞的立体纤毛，使用高度 精确的电子显微镜蛋白质定位技术。免疫金抗体标记的初步研究 结果在出生后第4天，我们将PKHD1L1定位于立体纤毛的顶端。在目标2中，我们检测了PKHD1L1的功能 通过评估毛细胞特异性PKHD1L1基因敲除小鼠的听力障碍。我们展示了“立体纤毛” 在PKHD1L1缺失的立体纤毛的顶端没有被毛。我们现在将特别关注其影响 PKHD1L1缺乏对被膜附着冠、束内聚、束发育的影响 刺激时的运动，以及毛细胞转导电流的特性。通过研究PKHD1L1- ，我们提出了一种干净的实验范式，以 测试PKHD1L1‘S(因此，涂层的)对束聚合力和滑动粘附力的贡献。最后，在我们的目标3中， 基于预测的PKHD1L1的结构域结构，暗示可能的同聚体(即与其自身)和 异构体(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 与其他参与形成覆盖膜或其附着体纤毛的蛋白质的相互作用，如 立体花青素、CEACAM16和ectorin。

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.